U.S. patent application number 12/489750 was filed with the patent office on 2009-12-24 for colchicine products, method of manufacture, and methods of use.
This patent application is currently assigned to MUTUAL PHARMACEUTICAL COMPANY, INC.. Invention is credited to Matthew W. Davis, Jie Du, Kurt R. Nielsen, Richard H. Roberts.
Application Number | 20090318561 12/489750 |
Document ID | / |
Family ID | 41431881 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090318561 |
Kind Code |
A1 |
Davis; Matthew W. ; et
al. |
December 24, 2009 |
COLCHICINE PRODUCTS, METHOD OF MANUFACTURE, AND METHODS OF USE
Abstract
Disclosed herein is a method of using colchicine. In one
embodiment, the method comprises administering to a patient
colchicine and a substrate of cytochrome P450 1A2 and monitoring
the patient during administration of colchicine and the substrate
for an adverse event. Also disclosed are articles of manufacture
comprising a container containing a dosage form of colchicine and a
method of manufacturing a colchicine product.
Inventors: |
Davis; Matthew W.; (Erwinna,
PA) ; Du; Jie; (Lansdale, PA) ; Nielsen; Kurt
R.; (Chadds Ford, PA) ; Roberts; Richard H.;
(Lakewood, NJ) |
Correspondence
Address: |
CANTOR COLBURN, LLP
20 Church Street, 22nd Floor
Hartford
CT
06103
US
|
Assignee: |
MUTUAL PHARMACEUTICAL COMPANY,
INC.
Philadelphia
PA
|
Family ID: |
41431881 |
Appl. No.: |
12/489750 |
Filed: |
June 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61074818 |
Jun 23, 2008 |
|
|
|
Current U.S.
Class: |
514/625 ;
705/2 |
Current CPC
Class: |
A61K 31/165 20130101;
A61P 19/06 20180101; G16H 70/40 20180101; G16H 40/63 20180101 |
Class at
Publication: |
514/625 ;
705/2 |
International
Class: |
A61K 31/165 20060101
A61K031/165; A61P 19/06 20060101 A61P019/06; G06Q 50/00 20060101
G06Q050/00 |
Claims
1. A method of determining risk of an adverse event in
administration of colchicine comprising determining for a patient
to whom colchicine is going to be administered or is being
administered whether a substance that is currently being or will be
administered to the patient is a substrate of CYP1A2; and
determining risk for the patient of an adverse event during
coadministration of colchicine and the substance resulting from
reduced metabolism of the substance by CYP1A2.
2. The method of claim 1, comprising determining risk for the
patient of an adverse event during coadministration of colchicine
and the substance resulting from reduced metabolism of the
substance by CYP1A2, wherein the reduced metabolism of the
substance by CYP1A2 is due to down-regulation of CYP1A2 expression
by colchicine.
3. The method of claim 1, wherein determining risk comprises
accessing a pharmacy management system.
4. The method of claim 1, further comprising administering
colchicine to the patient with the substance if there is not a risk
of an adverse event.
5. The method of claim 1, further comprising administering
colchicine to the patient with the substance if risk of an adverse
event is determined to be acceptable.
6. The method of claim 1, further comprising administering
colchicine to the patient but not administering the substance if
there is a risk of an adverse event.
7. The method of claim 1, comprising administering colchicine to
the patient but not administering the substance if there is an
unacceptable risk of an adverse event.
8. The method of claim 1, wherein the patient has gout or an attack
of acute gouty arthritis.
9.-10. (canceled)
11. A method of coadministration of colchicine and a substrate of
CYP1A2 to a patient comprising, administering colchicine and a
substrate of CYP1A2 to a patient in need of colchicine and the
substrate; monitoring the patient during coadministration of the
colchicine and the substrate; and adjusting the dosing of
colchicine or the substrate in response to the monitoring such that
an adverse event associated with the coadministration of colchicine
and a substrate of CYP1A2 is avoided.
12. The method of claim 11, wherein monitoring the patient
comprises monitoring the patient's plasma concentration of the
substrate; monitoring the patient for an adverse reaction
associated with elevated substrate plasma concentration; monitoring
the patient for a symptom of an active agent interaction between
the substrate and colchicine; monitoring the patient for an adverse
reaction resulting from coadministration of the substance and the
substrate; monitoring the patient for an adverse reaction or side
effect associated with the substrate; monitoring the patient for a
substrate-associated toxicity; or monitoring the patient for a
symptom of elevated plasma concentration of the substrate.
13. The method of claim 11, wherein the patient has acute gouty
arthritis; chronic gout; a cystic disease comprising polycystic
kidney disease or cystic fibrosis; a lentiviral infection; a
demyelinating disease of central or peripheral origin; multiple
sclerosis; cancer; an inflammatory disorder comprising rheumatoid
arthritis; glaucoma; Dupuytren's contracture; idiopathic pulmonary
fibrosis; primary amyloidosis; recurrent pericarditis; acute
pericarditis; asthma; postpericardiotomy syndrome; proliferative
vitreoretinopathy; Behcet's disease; Familial Mediterranean fever;
idiopathic thrombocytopenic purpura; primary biliary cirrhosis; or
pyoderma gangrenosum; or is in need of enhanced bone mineral
density.
14.-32. (canceled)
33. A method of administering colchicine to a patient in need
thereof, comprising receiving information that colchicine a) is
metabolized by cytochrome P450 2A6, 2B6, 2C8, 2C9 or 2C19; b)
inhibited cytochrome P450 2A6 or 2C8 enzyme activity in an in vitro
inhibition study; c) activated CYP3A4 enzyme activity in an in
vitro inhibition study; d) suppressed enzyme activity of cytochrome
P450 1A2, 2A6, 2C19, 2D6, or 2E1 in an in vitro induction study; or
e) suppressed mRNA expression of cytochrome P450 1A2 in an in vitro
induction study; and adjusting administration of colchicine and an
active agent to a patient in response to the information to avoid
an adverse event in the patient.
34. The method of claim 33, wherein the method further comprises
informing the patient or the patient's medical care worker that
administration of colchicine with an active agent that is a known
substrate of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 can result
in reduced metabolism of the active agent or increased plasma
concentration of the active agent; or monitoring the patient during
administration of colchicine.
35. The method of claim 34, wherein monitoring the patient
comprises: monitoring the patient's plasma concentration of the
active agent or colchicine; monitoring the patient for symptoms of
an active agent interaction between the active agent and
colchicine; monitoring the patient for an adverse event associated
with elevated plasma concentration of the active agent; or
monitoring the patient for an adverse reaction or side effect
resulting from coadministration of the active agent and
colchicine.
36.-45. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/074,818 filed Jun. 23, 2008, hereby
incorporated by reference in its entirety.
BACKGROUND
[0002] This application relates to colchicine products for
therapeutic purposes, and in particular to improved methods of use
of colchicine.
[0003] Colchicine is an alkaloid originally prepared from the dried
corms and seeds of Colchicum autumnale, the autumn crocus or meadow
saffron. The chemical name for colchicine is (S)N-(5, 6, 7,
9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[alpha]heptaien-7-yl)
acetamide. It is a pale yellow powder soluble in water in 1:25
dilution.
[0004] Colchicine is used for treatment and relief of pain and
other symptoms of attacks of acute gouty arthritis (also called
acute gouty flares or plainly, acute gout), which may include
swelling, redness and warmth. It is also recommended for regular
use between attacks as a prophylactic measure for chronic gout.
[0005] Colchicine is a microtubule-disrupting agent used in the
treatment of gout, particularly in the treatment of acute gouty
arthritis. Colchicine impairs the motility of granulocytes and can
prevent the inflammatory phenomena that initiate an attack of gout.
Colchicine also inhibits mitosis, thus affecting cells with high
turnover such as those in the gastrointestinal tract and bone
marrow; therefore, the primary side effects include
gastrointestinal upset such as diarrhea and nausea. Colchicine is
typically administered in 1- to 1.2-mg doses, with follow-up doses
of 0.5 to 0.6 mg twice daily. The beneficial effects of colchicine
in the treatment of acute gouty flares has traditionally taken up
to 48 hours to manifest; therefore, multi-dose therapy is likely
during the treatment of gout.
[0006] One of the most important groups of Phase I metabolic
enzymes are the cytochrome p450 monooxygenase system enzymes. The
cytochrome p450 enzymes are a highly diverse superfamily of
enzymes. NADPH is required as a coenzyme and oxygen is used as a
substrate. Each enzyme is termed an isoform or isozyme since each
derives from a different gene.
[0007] Many members of the cytochrome p450 family are known to
metabolize active agents in humans. Active agent interactions
associated with metabolism by cytochrome p450 isoforms generally
result from enzyme inhibition or enzyme induction. Enzyme
inhibition often involves competition between two active agents for
the substrate-binding site of the enzyme, although other mechanisms
for inhibition exist. Enzyme induction occurs when an active agent
activates an enzyme or stimulates the synthesis of more enzyme
protein, enhancing the enzyme's metabolizing capacity.
[0008] Cytochrome p450 isozymes identified as important in active
agent metabolism are CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Examples of cytochrome p450
enzymes known to be involved in active agent interactions are the
CYP3A subfamily, which is involved in many clinically significant
active agent interactions, including those involving non-sedating
antihistamines and cisapride, and CYP2D6, which is responsible for
the metabolism of many psychotherapeutic agents, such as
thioridazine. CYP1A2 and CYP2E1 enzyme are involved in active agent
interactions involving theophylline. CYP2C9, CYP1A2, and CYP2C19
are involved in active agent interactions involving warfarin.
Phenyloin and fosphenyloin are metabolized by CYP2C9, CYP2C19, and
CYP3A4.
[0009] Additionally, several cytochrome p450 isozymes are known to
be genetically polymorphic, leading to altered substrate
metabolizing ability in some individuals. Allelic variants of
CYP2D6 are the best characterized, with many resulting in an enzyme
with reduced, or no, catalytic activity. Gene duplication also
occurs. As a result, four phenotypic subpopulations of metabolizers
of CYP2D6 substrates exist: poor (PM), intermediate (IM), extensive
(EM), and ultrarapid (UM). The genetic polymorphisms vary depending
on the population in question. For example, Caucasian populations
contain a large percentage of individuals who are poor
metabolizers, due to a deficiency in CYP2D6--perhaps 5-10% of the
population, while only 1-2% of Asians are PMs. CYP2C9, which
catalyzes the metabolism of a number of commonly used active
agents, including that of warfarin and phenyloin, is also
polymorphic. The two most common CYP2C9 allelic variants have
reduced activity (5-12%) compared to the wild-type enzyme. Genetic
polymorphism also occurs in CYP2C19, for which at least 8 allelic
variants have been identified that result in catalytically inactive
protein. About 3% of Caucasians are poor metabolizers of active
agents metabolized by CYP2C19, while 13-23% of Asians are poor
metabolizers of active agents metabolized by CYP2C19. Allelic
variants of CYP2A6 and CYP2B6 have also been identified as
affecting enzyme activity. At least one inactive CYP2A6 variant
occurs in Caucasians at a frequency of 1-3%, resulting in a PM
phenotype. A whole gene deletion has been identified in a Japanese
population, with an allelic frequency of 21%; homozygotes in this
mutation show a PM phenotype. For CYP2B6, about 3-4% of Caucasians
have a polymorphism producing a PM phenotype.
[0010] Tateishi et al. (Biochem. Pharmacol. (1997) 53:111-116)
studied the biotransformation of colchicine in human liver
microsomes in order to identify particular human cytochrome P450
isozymes responsible for the formation of its demethylated
metabolites. Formation of 3-demethylchochicine and
2-demethylcolchicine was correlated with CYP3A4 activity, but not
with activity of CYP2A6, CYP2C9, CYP2C19, CYP2D6, or CYP2E1.
Metabolism of colchicine by CYP3A4 was confirmed by using
antibodies against CYP3A4 and chemical inhibition of CYP3A4.
[0011] Studies on the effect of colchicine on expression of
selected cytochrome P450 isozymes in primary cultures of human
hepatocytes have also been published. Dvorak et al. (Acta Univ.
Palacki. Olomuc., Fac. Med. (2000) 143:47-50) provided preliminary
data on the effect of colchicine and several of its derivatives on
protein levels of CYP1A2, CYP2A6, CYP2C9/19, CYP2E1, and CYP3A4 by
immunoblotting. Colchicine caused an increase in CYP2E1 protein
levels and appeared to decrease protein levels of CYP1A2,
CYP2C9/19, and CYP3A4, with 10 .mu.M colchicine causing a greater
reduction in each isozyme than 1 .mu.M colchicine. The colchicine
metabolite 3-demethylchochicine caused a decrease in protein for
CYP1A2, CYP2C9/19, CYP2E1, and CYP3A4. The levels of CYP2A6 were
unaffected by colchicine or any of the tested metabolites. In a
more complete report on expression of CYP1A2, CYP2A6, CYP2C9,
CYP2C19, CYP2E1, and CYP3A4 in primary cultures of human
hepatocytes, Dvorak et al. (Toxicology in Vitro (2002) 16:219-227)
concluded that CYP1A2 protein content in 1 .mu.M colchicine treated
cells was not different from that in control cells, while the
inducer TCDD increased the level of CYP1A2 protein by an average of
three-fold. The levels of CYP2A6 protein were also unaffected by
colchicine. The enzyme activities of CYP3A4 and CYP2C9 were
significantly decreased by colchicine, whereas activity of CYP2E1
was not affected. Northern blots showed that colchicine suppressed
CYP2C9 mRNA levels by about 20% and did not alter CYP3A4 mRNA
levels as compared to control cells. A subsequent study by Dvorak
et al. (Mol. Pharmacol. (2003) 64:160-169) showed that colchicine
decreased both basal and rifampicin-inducible and
phenobarbital-inducible expression of CYP2B6, CYP2C8/9, and
CYP3A4.
[0012] Active agent interactions present a health risk to patients
and a medical challenge for all medical care workers. Various
studies of adverse reactions from exposure to active agents have
found that 6.5-23% of the adverse reactions result from active
agent interactions. Unfortunately, each year a number of deaths
occur as the direct result of patients taking a new prescription
pharmaceutical product in combination with their existing
medication regimen. By understanding the unique functions and
characteristics of Phase I and Phase II metabolic enzymes, such as
the cytochrome p450 enzyme superfamily, medical care workers such
as physicians and pharmacists may better avoid or safely manage
active agent interactions and may better anticipate or explain an
individual's response to a particular therapeutic regimen.
[0013] There accordingly remains a need in the art for improved
methods for the administration and use of colchicine, in particular
methods that take into account the effects of colchicine on
metabolism by cytochrome P450 isozymes.
SUMMARY
[0014] Disclosed herein are methods of using colchicine. Colchicine
can be used in prevention or treatment of various diseases or
conditions, including, for example, attacks of acute gouty
arthritis and pain and other symptoms in attacks of acute gouty
arthritis, chronic gout (prophylaxis of gouty arthritis), a cystic
disease, for example polycystic kidney disease or cystic fibrosis,
a lentiviral infection, demyelinating diseases of central or
peripheral origin, multiple sclerosis, cancer, an inflammatory
disorder such as rheumatoid arthritis, glaucoma, Dupuytren's
contracture, idiopathic pulmonary fibrosis, primary amyloidosis,
recurrent pericarditis, acute pericarditis, asthma,
postpericardiotomy syndrome, proliferative vitreoretinopathy,
Behcet's disease, Familial Mediterranean fever, idiopathic
thrombocytopenic purpura, primary biliary cirrhosis, and pyoderma
gangrenosum, or in enhancing bone formation or bone mineral
density.
[0015] In an embodiment, the method comprises administering
colchicine and a substance that is a substrate of cytochrome P450
1A2, 2A6, 2C19, 2D6, or 2E1 to a patient; and monitoring the
patient during administration of colchicine and the substance.
[0016] In an embodiment, the method comprises administering
colchicine to a patient in need thereof; determining that the
patient is taking a substance that is a substrate of cytochrome
P450 1A2, 2A6, 2C19, 2D6, or 2E1; and adjusting administration of
colchicine or the substance to the patient to avoid an adverse
reaction.
[0017] In an embodiment, the method comprises determining that a
patient in need colchicine therapy is taking a substance that is a
substrate of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1, and
adjusting administration to the patient of colchicine or the
substance to avoid an adverse event associated with suppression of
metabolism of the substance by colchicine.
[0018] In an embodiment, the method comprises administering
colchicine and a substrate of cytochrome P450 1A2, 2A6, 2C19, 2D6,
or 2E1 to a patient; and altering dosing of the substrate or
colchicine for the patient if substrate plasma concentration of the
patient increases during coadministration with colchicine.
[0019] In an embodiment, the method comprises administering
colchicine and a substrate of cytochrome P450 1A2, 2A6, 2C19, 2D6,
or 2E1 to a patient; determining that the patient experiences a
substrate-associated toxicity during coadministration with
colchicine; and altering dosing of the substrate or colchicine such
that the substrate-associated toxicity is reduced.
[0020] In an embodiment, the method comprises administering
colchicine to a patient in need of colchicine therapy; determining
that a substance that is a substrate of cytochrome P450 1A2, 2A6,
2C19, 2D6, or 2E1 is administered to the patient; and monitoring
the patient during administration of colchicine and the
substance.
[0021] In an embodiment, the method comprises determining for a
patient to whom colchicine is going to be administered or is being
administered whether a substance that is currently being or will be
administered to the patient is a substrate of a cytochrome P450 1A2
(CYP1A2); and determining risk for the patient of an adverse event
resulting from reduced metabolism of the substance by CYP1A2 during
coadministration of colchicine and the substance.
[0022] In an embodiment, the method comprises determining a dosing
regimen for a substrate of cytochrome P450 1A2 to be administered
to a patient in need thereof; determining that colchicine is
administered to the patient, and altering the determined dosing
regimen of the substrate during coadministration of colchicine to
prevent a substrate-associated toxicity.
[0023] In an embodiment, the method comprises informing a user that
colchicine is metabolized by cytochrome P450 2A6, 2B6, 2C8, 2C9 or
2C19; inhibited cytochrome P450 2A6 or 2C8 enzyme activity in an in
vitro inhibition study; activated CYP3A4 enzyme activity in an in
vitro inhibition study; suppressed enzyme activity of cytochrome
P450 1A2, 2A6, 2C19, 2D6, or 2E1 in an in vitro induction study; or
suppressed mRNA expression of cytochrome P450 1A2 in an in vitro
induction study.
[0024] In an embodiment, the method comprises obtaining colchicine
from a container associated with published material providing
information that colchicine is metabolized by cytochrome P450 2A6,
2B6, 2C8, 2C9 or 2C19; inhibited cytochrome P450 2A6 or 2C8 enzyme
activity in an in vitro inhibition study; activated CYP3A4 enzyme
activity in an in vitro inhibition study; suppressed enzyme
activity of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 in an in
vitro induction study; or suppressed mRNA expression of cytochrome
P450 1A2 in an in vitro induction study.
[0025] Also disclosed herein are methods of manufacturing a
colchicine product.
[0026] In one embodiment, the method comprises packaging a
colchicine dosage form with published material providing
information that colchicine is metabolized by cytochrome P450 2A6,
2B6, 2C8, 2C9 or 2C19; inhibited cytochrome P450 2A6 or 2C8 enzyme
activity in an in vitro inhibition study; activated CYP3A4 enzyme
activity in an in vitro inhibition study; suppressed enzyme
activity of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 in an in
vitro induction study; or suppressed mRNA expression of cytochrome
P450 1A2 in an in vitro induction study.
[0027] Also disclosed herein are articles of manufacture comprising
a container containing a dosage form of colchicine.
[0028] In one embodiment, the container is associated with
published material informing that colchicine is metabolized by
cytochrome P450 2A6, 2B6, 2C8, 2C9 or 2C19; inhibited cytochrome
P450 2A6 or 2C8 enzyme activity in an in vitro inhibition study;
activated CYP3A4 enzyme activity in an in vitro inhibition study;
suppressed enzyme activity of cytochrome P450 1A2, 2A6, 2C19, 2D6,
or 2E1 in an in vitro induction study; or suppressed mRNA
expression of cytochrome P450 1A2 in an in vitro induction
study
[0029] These and other embodiments, advantages and features of the
present invention become clear when detailed description and
examples are provided in subsequent sections.
DETAILED DESCRIPTION
[0030] Disclosed herein are methods of using colchicine and
colchicine products. The inventors have determined certain effects
of colchicine on the activity of various cytochrome P450 isozymes
and identified risks associated with administration of colchicine
with another substance resulting from these effects of colchicine
on the activity of the cytochrome P450 isozymes. With the knowledge
of the particular information, a medical care worker can better
avoid or safely manage an active agent interaction in a patient
between colchicine and the substance, and its resultant effects on
efficacy or safety of colchicine or the substance. Specifically,
knowledge of the particular information permits the administration
of colchicine or the substance to be optimized for the patient by a
medical care worker to provide safe use of colchicine or the
substance, while oftentimes reducing or minimizing side effects or
adverse events resulting from the effects. Knowledge of the
particular information permits a medical care worker to use
colchicine to treat a patient that is taking another substance more
effectively and with fewer risks by allowing proper dosing,
dispensing, and administration of colchicine or the substance to
the patient by the patient's medical care worker to avoid, or
reduce risk of occurrence of a sub-therapeutic effect, a side
effect, an adverse reaction, or an active agent interaction between
colchicine and the substance and alerts the patient and the
patient's medical care worker to the need to monitor the patient
for symptoms of a sub-therapeutic effect, a side effect, an adverse
reaction, or an active agent interaction between colchicine and the
substance.
[0031] Enzymes involved in Phase I and Phase II active agent
metabolism, such as the cytochrome p450 isozymes, respond to the
constantly changing types and amounts of substrates they encounter.
For example, changes in active agent metabolism due to competition
for the same cytochrome P450 isoform can change the clinical
effectiveness or safety of an active agent by altering the plasma
concentration of the active agent or its metabolite(s). Similarly,
inhibition or induction of the cytochrome P450 isoform that
metabolizes a particular active agent can change the clinical
effectiveness or safety of that active agent. For the case in which
the active agent is a narrow therapeutic index active agent, such
as warfarin or phenyloin, too little of the active agent in the
blood stream can lead to insufficient therapeutic activity, while a
too large dose of the active agent can lead to excessive
therapeutic activity or toxicity, either of which can be
detrimental to the patient. For example, since colchicine
down-regulates CYP1A2 mRNA expression and enzyme activity in
primary cultures of human hepatocytes, the administration of
colchicine with a substance that is a substrate of CYP1A2 can
decrease the metabolism by CYP1A2 of that substrate.
[0032] Colchicine therapy can be considered optimal when effective
plasma levels are reached when required. In addition, peak plasma
values (C.sub.max) should be as low as possible so as to reduce the
incidence and severity of possible side effects.
[0033] The terms "a" and "an" do not denote a limitation of
quantity, but rather denote the presence of at least one of the
referenced item. The term "or" means "and/or". The terms
"comprising", "having", "including", and "containing" are to be
construed as open-ended terms (i.e., meaning "including, but not
limited to").
[0034] An "active agent" means a compound (including for example,
colchicine), element, or mixture that when administered to a
patient, alone or in combination with another compound, element, or
mixture, confers, directly or indirectly, a physiological effect on
the patient. The indirect physiological effect may occur via a
metabolite or other indirect mechanism. When the active agent is a
compound, then salts, solvates (including hydrates), and
co-crystals of the free compound or salt, crystalline forms,
non-crystalline forms, and any polymorphs of the compound are
contemplated herein. Compounds may contain one or more asymmetric
elements such as stereogenic centers, stereogenic axes and the
like, e.g., asymmetric carbon atoms, so that the compounds can
exist in different stereoisomeric forms. These compounds can be,
for example, racemates or optically active forms. For compounds
with two or more asymmetric elements, these compounds can
additionally be mixtures of diastereomers. For compounds having
asymmetric centers, all optical isomers in pure form and mixtures
thereof are encompassed. In addition, compounds with carbon-carbon
double bonds may occur in Z- and E-forms, with all isomeric forms
of the compounds. In these situations, the single enantiomers,
i.e., optically active forms can be obtained by asymmetric
synthesis, synthesis from optically pure precursors, or by
resolution of the racemates. Resolution of the racemates can also
be accomplished, for example, by conventional methods such as
crystallization in the presence of a resolving agent, or
chromatography, using, for example a chiral HPLC column. All forms
are contemplated herein regardless of the methods used to obtain
them.
[0035] All forms (for example solvates, optical isomers,
enantiomeric forms, polymorphs, free compound, and salts) of
colchicine or other active agent may be employed either alone or in
combination.
[0036] "Active agent interaction" refers to a change in the
metabolism or the pharmacology of an active agent in a patient that
can occur with co-administration of a second active agent. A
"potential active agent interaction" refers to an active agent
interaction between two active agents that is theoretically
possible based on knowledge that one of the active agents is
metabolized by a given cytochrome p450 isozyme and that the second
of the active agents is a substrate, inhibitor, or inducer of that
cytochrome p450 isozyme.
[0037] "Administering colchicine with a substance", "administering
colchicine and a substance", or "co-administering colchicine and a
substance" means colchicine and the substance are administered
simultaneously in a single dosage form, administered concomitantly
in separate dosage forms, or administered in separate dosage forms
separated by some amount of time that is within the time in which
both colchicine and the substance are within the blood stream of a
patient. The colchicine and the substance need not be prescribed
for a patient by the same medical care worker. The substance need
not require a prescription. Administration of colchicine or the
substance can occur via any appropriate route, for example, oral
tablets, oral capsules, oral liquids, inhalation, injection,
suppositories, or topical contact.
[0038] "Adverse event" means any untoward medical occurrence in a
patient administered an active agent and which does not necessarily
have to have a causal relationship with this treatment. An adverse
event (AE) can therefore be any unfavorable and unintended sign
(including an abnormal laboratory finding, for example), symptom,
or disease temporally associated with the use of the active agent,
whether or not considered related to the active agent.
[0039] "Adverse reaction" means a response to an active agent which
is noxious and unintended and which occurs at doses normally used
in humans for prophylaxis, diagnosis, or therapy of disease or for
modification of physiological function. The unintended response can
be an unexpected diminished or enhanced pharmacologic activity or
toxicity of the active agent, e.g., a colchicine-associated
toxicity. An adverse reaction also includes any undesirable or
unexpected event requiring discontinuation of the active agent,
modification of the dose, prolonged hospitalization, or the
administration of supportive treatment.
[0040] "Affects" include an increase or decrease in degree, level,
or intensity; a change in time of onset or duration; a change in
type, kind, or effect, or a combination comprising at least one of
the foregoing.
[0041] As used herein, "allelic variant" means one of the
alternative forms at a genetic locus on a single chromosome. For
loci in most of the human genome, a human has two chromosomes,
which may carry the same or two different allelic variants.
[0042] "Adjusting administration of an active agent", "altering
administration of an active agent", or "altering" or "adjusting"
dosing of an active agent are all equivalent and mean making no
change in the dose or dosing regimen of the active agent; tapering
off, reducing or increasing the dose or the interval between doses
of the active agent, ceasing to administer the active agent to the
patient, or substituting a different active agent for the active
agent.
[0043] "Dosing regimen" means the dose of an active agent taken at
a first time by a patient and the interval (time or symptomatic) at
which any subsequent doses of the active agent are taken by the
patient. The additional doses of the active agent can be different
from the dose taken at the first time.
[0044] A "dose" means the measured quantity of an active agent to
be taken at one time by a patient.
[0045] "Bioavailability" means the extent or rate at which an
active agent is absorbed into a living system or is made available
at the site of physiological activity. For active agents that are
intended to be absorbed into the bloodstream, bioavailability data
for a given formulation may provide an estimate of the relative
fraction of the administered dose that is absorbed into the
systemic circulation. "Bioavailability" can be characterized by one
or more pharmacokinetic parameters.
[0046] A "dosage form" means a unit of administration of an active
agent. Examples of dosage forms include tablets, capsules,
injections, suspensions, liquids, emulsions, creams, ointments,
suppositories, inhalable forms, transdermal forms, and the
like.
[0047] The term "effective amount" or "therapeutically effective
amount" means an amount effective, when administered to a patient,
to provide any therapeutic benefit. A therapeutic benefit may be an
amelioration of symptoms, e.g., an amount effective to decrease the
symptoms of acute gouty arthritis, for example pain associated with
an attack of acute gouty arthritis. The amount that is "effective"
will vary from subject to subject, depending on the age and general
condition of the individual, the particular active agent, and the
like. Thus, it is not always possible to specify an exact
"effective amount." However, an appropriate "effective" amount in
any individual case may be determined by one of ordinary skill in
the art using routine experimentation. In certain circumstances a
patient may not present symptoms of a condition for which the
patient is being treated. A therapeutically effective amount of an
active agent may also be an amount sufficient to provide a
significant positive effect on any indicium of a disease, disorder,
or condition, e.g. an amount sufficient to significantly reduce the
severity of an attack of acute gouty arthritis. A significant
effect on an indicium of a disease, disorder, or condition is
statistically significant in a standard parametric test of
statistical significance, for example Student's T-test, where
p.ltoreq.0.05. An "effective amount" or "therapeutically effective
amount" of colchicine may also be an amount of about 10 mg per day
or less, specifically about 8 mg per day or less, or of any dosage
amount approved by a governmental authority such as the United
States Food and Drug Administration (FDA), for use in treatment.
For example, an effective amount can be up to 4.8 mg colchicine per
incident of acute gout, or 0.5 or 0.6 mg colchicine twice daily for
either prophylaxis of chronic gout or treatment of Behcet's disease
or Familial Mediterranean fever. In some embodiments amounts of 8
mg colchicine per day, 1.0 or 1.2 mg colchicine per unit dosage
form, or 0.5 or 0.6 mg colchicine or less per unit dosage form is
an "effective amount" or "therapeutically effective amount" of
colchicine.
[0048] "Efficacy" means the ability of an active agent administered
to a patient to produce a therapeutic effect in the patient.
[0049] "Enhancing the safety profile" of an active agent means
implementing actions or articles designed or intended to help
reduce the incidence of adverse events associated with
administration of the active agent, including adverse effects
associated with patient-related factors (e.g., age, gender,
ethnicity, race, target illness, abnormalities of renal or hepatic
function, co-morbid illnesses, genetic characteristics such as
metabolic status, or environment) and active agent-related factors
(e.g., dose, plasma level, duration of exposure, or concomitant
medication).
[0050] "Informing" means referring to or providing published
material, for example, providing an active agent with published
material to a user; or presenting information orally, for example,
by presentation at a seminar, conference, or other educational
presentation, by conversation between a pharmaceutical sales
representative and a medical care worker, or by conversation
between a medical care worker and a patient; or demonstrating the
intended information to a user for the purpose of
comprehension.
[0051] "Labeling" means all labels or other means of written,
printed, graphic, electronic, verbal, or demonstrative
communication that is upon a dosage form or packaging of a
pharmaceutical product or that accompanies a dosage form in a
pharmaceutical product.
[0052] A "medical care worker" means a worker in the health care
field who may need or utilize information regarding an active
agent, including a dosage form thereof, including information on
safety, efficacy, dosing, administration, or pharmacokinetics.
Examples of medical care workers include physicians, pharmacists,
physician's assistants, nurses, aides, caretakers (which can
include family members or guardians), emergency medical workers,
and veterinarians.
[0053] As used herein, an enzyme "metabolizing" a substance means
the substance is a substrate of the enzyme, i.e., the enzyme can
chemically transform the substance.
[0054] A substance having a "narrow therapeutic index" (NTI) means
a substance falling within any definition of narrow therapeutic
index as promulgated by the U.S. Food and Drug Administration or
any successor agency thereof. For example, a substance having a
"narrow therapeutic index" can be a substance having a less than
2-fold difference in median lethal dose (LD50) and median effective
dose (ED50) values or having a less than 2-fold difference in the
minimum toxic concentration and minimum effective concentration in
the blood; and for which safe and effective use of the substance
requires careful titration and patient monitoring.
[0055] "Oral dosage form" includes a dosage form for oral
administration.
[0056] A "patient" means a human or non-human animal in need of
medical treatment. Medical treatment can include treatment of an
existing condition, such as a disease or disorder, prophylactic or
preventative treatment, or diagnostic treatment. In some
embodiments the patient is a human patient.
[0057] A "pharmaceutical supplier" means a person (other than a
medical care worker), business, charitable organization,
governmental organization, or other entity involved in the transfer
of active agent, including a dosage form thereof, between entities,
for profit or not. Examples of pharmaceutical suppliers include
pharmaceutical distributors, pharmaceutical wholesalers,
pharmaceutical benefits managers, pharmacy chains, pharmacies
(online or physical), hospitals, HMOs, supermarkets, the Veterans
Administration, or foreign businesses or individuals importing
active agent into the United States.
[0058] "Pharmacokinetic parameters" describe the in vivo
characteristics of an active agent (or surrogate marker for the
active agent) over time, such as plasma concentration (C),
C.sub.min, C.sub.max, C.sub.n, C.sub.24, T.sub.max, and AUC.
"C.sub.max" is the measured concentration of the active agent in
the plasma at the point of maximum concentration. "C.sub.min" is
the measured concentration of the active agent in the plasma at the
point of minimum concentration at steady state. "C.sub.n" is the
measured concentration of an active agent in the plasma at about n
hours after administration. "C.sub.24" is the measured
concentration of an active agent in the plasma at about 24 hours
after administration. The term "T.sub.max" refers to the time at
which the measured concentration of an active agent in the plasma
is the highest after administration of the active agent. "AUC" is
the area under the curve of a graph of the measured concentration
of an active agent (typically plasma concentration) vs. time,
measured from one time point to another time point. For example
AUC.sub.0-t is the area under the curve of plasma concentration
versus time from time 0 to time t. The AUC.sub.0-.infin. or
AUC.sub.0-INF is the calculated area under the curve of plasma
concentration versus time from time 0 to time infinity.
[0059] "Pharmaceutically acceptable salts" include derivatives of
the active agent (e.g., colchicine), wherein the parent compound is
modified by making acid or base addition salts thereof, and further
refers to pharmaceutically acceptable solvates, including hydrates,
and co-crystals of such compounds and such salts. All forms of such
derivatives of colchicine are contemplated herein, including all
crystalline, amorphous, and polymorph forms. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid addition salts; and the like, and
combinations comprising one or more of the foregoing salts. The
pharmaceutically acceptable salts include salts, for example, from
inorganic or organic acids. For example, acid salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric and the like.
Pharmaceutically acceptable organic salts includes salts prepared
from organic acids such as acetic, trifluoroacetic, propionic,
succinic, glycolic, stearic, lactic, malic, tartaric, citric,
ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,
benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, HOOC--(CH.sub.2).sub.n--COOH where
n is 0-4, and the like; organic amine salts such as triethylamine
salt, pyridine salt, picoline salt, ethanolamine salt,
triethanolamine salt, dicyclohexylamine salt, N,N'
dibenzylethylenediamine salt, and the like; and amino acid salts
such as arginate, asparginate, glutamate, and the like; and
combinations comprising one or more of the foregoing salts.
Specific colchicine salts include colchicine hydrochloride,
colchicine dihydrochloride, and co-crystals, hydrates or solvates
thereof.
[0060] "Phenotype" means an observable trait of an organism
resulting from the interplay of environment and genetics. Examples
include apparent rate of metabolism of substrates by a cytochrome
p450 isozyme of an organism, such as the "poor metabolizer" (PM) or
"ultrarapid metabolizer" (UM) phenotypes identified in humans for
metabolism of substrates metabolized by CYP2D6.
[0061] A "product" or "pharmaceutical product" means a dosage form
of an active agent plus published material, and optionally
packaging.
[0062] "Providing" means giving, administering, selling,
distributing, transferring (for profit or not), manufacturing,
compounding, or dispensing.
[0063] "Published material" means a medium providing information,
including printed, audio, visual, or electronic medium, for example
a flyer, an advertisement, a product insert, printed labeling, an
internet web site, an internet web page, an internet pop-up window,
a radio or television broadcast, a compact disk, a DVD, an audio
recording, or other recording or electronic medium.
[0064] "Product insert" means the professional labeling
(prescribing information) for a pharmaceutical product, a patient
package insert for the pharmaceutical product, or a medication
guide for the pharmaceutical product.
[0065] "Professional labeling" or "prescribing information" means
the official description of a pharmaceutical product approved by a
regulatory agency (e.g., FDA or EMEA) regulating marketing of the
pharmaceutical product, which includes a summary of the essential
scientific information needed for the safe and effective use of the
drug, such as, for example indication and usage; dosage and
administration; who should take it; adverse events (side effects);
instructions for use in special populations (pregnant women,
children, geriatric, etc.); safety information for the patient, and
the like.
[0066] "Patient package insert" means information for patients on
how to safely use a pharmaceutical product that is part of the
FDA-approved labeling. It is an extension of the professional
labeling for a pharmaceutical product that may be distributed to a
patient when the product is dispensed which provides
consumer-oriented information about the product in lay language,
for example it may describe benefits, risks, how to recognize
risks, dosage, or administration.
[0067] "Medication Guide" means an FDA-approved patient labeling
for a pharmaceutical product conforming to the specifications set
forth in 21 CFR 208 and other applicable regulations which contains
information for patients on how to safely use a pharmaceutical
product. A medication guide is scientifically accurate and is based
on, and does not conflict with, the approved professional labeling
for the pharmaceutical product under 21 CFR 201.57, but the
language need not be identical to the sections of approved labeling
to which it corresponds. A medication guide is typically available
for a pharmaceutical product with special risk management
information.
[0068] As used herein, "colchicine therapy" refers to medical
treatment of a symptom, disorder, or condition by administration of
colchicine.
[0069] "Risk" means the probability or chance of adverse reaction,
injury, or other undesirable outcome arising from a medical
treatment. An "acceptable risk" means a measure of the risk of
harm, injury, or disease arising from a medical treatment that will
be tolerated by an individual or group. Whether a risk is
"acceptable" will depend upon the advantages that the individual or
group perceives to be obtainable in return for taking the risk,
whether they accept whatever scientific and other advice is offered
about the magnitude of the risk, and numerous other factors, both
political and social. An "acceptable risk" of an adverse reaction
means that an individual or a group in society is willing to take
or be subjected to the risk that the adverse reaction might occur
since the adverse reaction is one whose probability of occurrence
is small, or whose consequences are so slight, or the benefits
(perceived or real) of the active agent are so great. An
"unacceptable risk" of an adverse reaction means that an individual
or a group in society is unwilling to take or be subjected to the
risk that the adverse reaction might occur upon weighing the
probability of occurrence of the adverse reaction, the consequences
of the adverse reaction, and the benefits (perceived or real) of
the active agent. "At risk" means in a state or condition marked by
a high level of risk or susceptibility. Risk assessment consists of
identifying and characterizing the nature, frequency, and severity
of the risks associated with the use of a product.
[0070] "Safety" means the incidence or severity of adverse events
associated with administration of an active agent, including
adverse effects associated with patient-related factors (e.g., age,
gender, ethnicity, race, target illness, abnormalities of renal or
hepatic function, co-morbid illnesses, genetic characteristics such
as metabolic status, or environment) and active agent-related
factors (e.g., dose, plasma level, duration of exposure, or
concomitant medication).
[0071] A "sensitive plasma concentration profile active agent"
means an active agent for which a moderate change in plasma
concentration can have a deleterious effect on the prescribed
therapeutic intent.
[0072] "Side effect" means a secondary effect resulting from taking
an active agent. The secondary effect can be a negative
(unfavorable) effect or a positive (favorable) effect.
[0073] Solid dosage forms of colchicine comprise up to about 10 mg
colchicine, specifically about 0.25 to about 8 mg colchicine, more
specifically about 0.5 to about 4 mg colchicine, yet more
specifically about 0.5 to about 1.2 mg colchicine. In an
embodiment, solid dosage forms of colchicine comprise about 0.5 to
about 0.6 mg colchicine. Amounts in dosage forms are given for
colchicine free base, however equivalent amounts of other forms of
colchicine can be used. In one embodiment, the solid dosage form is
an oral dosage form, for example, a tablet.
[0074] A "substance" taken or administered with colchicine means a
substance that affects the safety, bioavailability, plasma
concentration, efficacy, or a combination comprising at least one
of the foregoing of colchicine or the substance. A "substance" can
be an active agent, an herbal supplement, a nutritional supplement,
a vitamin, a xenobiotic, or an environmental contaminant.
[0075] A substance is a "substrate" of enzyme activity when it can
be chemically transformed by action of the enzyme on the substance.
"Enzyme activity" refers broadly to the specific activity of the
enzyme (i.e., the rate at which the enzyme transforms a substrate
per mg or mole of enzyme) as well as to the metabolic effect of
such transformations. Thus, a substance is an "inhibitor" of enzyme
activity when the specific activity or the metabolic effect of the
specific activity of the enzyme can be decreased by the presence of
the substance, without reference to the precise mechanism of such
decrease. For example a substance can be an inhibitor of enzyme
activity by competitive, non-competitive, allosteric or other type
of enzyme inhibition, or other direct or indirect mechanisms.
Similarly, a substance is an "activator" of enzyme activity when
the specific activity or the metabolic effect of the specific
activity of the enzyme can be increased by the presence of the
substance, without reference to the precise mechanism of such
increase. For example a substance can be an activator of enzyme
activity by increasing reaction rate, by allosteric activation or
other direct or indirect mechanisms. Any of these effects on enzyme
activity can occur at a given concentration of active agent in a
single sample, donor, or patient without regard to clinical
significance. It is possible for a substance to be a substrate,
inhibitor, or activator of an enzyme activity. For example, the
substance can be an inhibitor of enzyme activity by one mechanism
and an activator of enzyme activity by another mechanism. The
function (substrate, inhibitor, or activator) of the substance with
respect to activity of an enzyme can depend on environmental
conditions.
[0076] A substance is a "suppressor" of observed enzyme activity in
an in vitro induction study when the measured activity per unit
number of cells is decreased by the presence of the substance,
without reference to the precise mechanism of such decrease. For
example, a substance can be a suppressor of enzyme activity in an
induction study by decreasing specific activity of a fixed amount
of enzyme or by decreasing enzyme level per cell for example by
decreasing translation of the enzyme's mRNA or by decreasing
transcription of the enzyme's gene, or by other direct or indirect
mechanisms for decreasing measured enzyme activity per unit number
of cells. A substance is an "inducer" of observed enzyme activity
in an in vitro induction study when the measured activity per unit
number of cells can be increased by the presence of the substance,
without reference to the precise mechanism of such increase. For
example, a substance can be an inducer of enzyme activity in the
induction study by increasing specific activity of a fixed amount
of enzyme, by increasing enzyme level per cell for example by
increasing translation of the enzyme's mRNA or increasing
transcription of the enzyme's gene, or by other direct or indirect
mechanisms for increasing measured enzyme activity per unit number
of cells.
[0077] A substance is a "suppressor" of enzyme expression in an in
vitro induction study when the expression of the gene of the enzyme
can be decreased by the presence of the substance, without
reference to the precise mechanism of such decrease. For example, a
substance can be a suppressor of enzyme expression by decreasing
translation of the enzyme's mRNA, by decreasing transcription of
the enzyme's gene, or other direct or indirect mechanisms for
decreasing expression of the enzyme. A substance is an "inducer" of
enzyme expression in an in vitro induction study when the
expression of the gene of the enzyme can be increased by the
presence of the substance, without reference to the precise
mechanism of such increase. For example, a substance can be an
inducer of enzyme expression by increasing translation of the
enzyme's mRNA, by increasing transcription of the enzyme's gene, or
other direct or indirect mechanisms for increasing expression of
the enzyme.
[0078] The function (suppressor or inducer) of the substance in an
in vitro induction study with respect to measured enzyme activity
or expression of the gene of an enzyme can depend on environmental
conditions.
[0079] A "strongly significant" result from an in vitro study means
a result which is a strong indicator of a potential in vivo
interaction between an active agent and another co-administered
substance. In vivo evaluation of the potential interaction between
the active agent and another co-administered substance can be
warranted to determine whether the interaction is sufficiently
large to necessitate a dosage adjustment of one or both substances,
or whether the interaction would require additional therapeutic
monitoring.
[0080] For an in vitro study, a strongly significant level of
observed induction by the active agent of a cytochrome p450 isozyme
means induction that is at least 40% of the change in induction
observed for a positive control inducer of the cytochrome p450
isozyme or at least a two-fold induction of the cytochrome p450
isozyme. Specifically, for a study using cultured primary
hepatocytes, this level of induction is obtained in samples from a
majority of the donors tested. More specifically, this level of
induction is obtained using a concentration of the active agent in
the range of plasma concentrations observed in vivo after
administration of the active agent or the level of observed
induction shows a concentration dependent trend in the samples of
each donor showing at least 40% of the change in induction observed
for a positive control inducer or at least a two-fold induction of
the cytochrome p450 isozyme.
[0081] Additionally, for an in vitro study, a strongly significant
level of observed inhibition of a cytochrome p450 isozyme by the
active agent means that the active agent reduced the activity of
the enzyme by 50% or more. Specifically, reduction in activity is
observed to occur in a dose dependent way to produce this level of
inhibition. More specifically, this level of reduction is obtained
at a concentration of the active agent in the range of plasma
concentrations observed in vivo after administration of the active
agent. Yet more specifically, when primary cultures of hepatocytes
are used in the enzyme activity assay, the level of reduction is
observed in the samples from a majority of the donors tested.
[0082] "Subtherapeutic outcome" means a response to an active agent
that is less than that anticipated from a dosing regimen of the
active agent used for treatment of disease or for modification of
physiological function.
[0083] The terms "treating" and "treatment" mean implementation of
therapy with the intention of reducing in severity or frequency
symptoms, elimination of symptoms or underlying cause, prevention
of the occurrence of symptoms or their underlying cause, or
improvement or remediation of damage.
[0084] A "user" means a patient, a medical care worker, or a
pharmaceutical supplier.
[0085] The cytochrome p450 enzymes are a highly diverse superfamily
of enzymes. Each cytochrome p450 enzyme is termed an "isoform" or
"isozyme" since each derives from a different gene. Cytochrome p450
enzymes are categorized into families and subfamilies by amino acid
sequence similarities. These enzymes are designated by the letters
"CYP" followed by an Arabic numeral representing the family, a
letter representing the sub-family and another Arabic numeral
representing a specific gene (e.g., CYP2D6). Particular isozymes
discussed herein are named as per the recommendations of the P450
Gene Superfamily Nomenclature Committee (see e.g., "P450
superfamily: Update on new sequences, gene mapping, accession
numbers, and nomenclature" Pharmacogenetics 6, 1-42 1996, part A
pp. 1-21). Herein, the designation for a cytochrome p450 isozyme
may encompass the homolog from any species identified as having
such an isozyme. For example, CYP1A2 genes are known in at least
rat, human, rabbit, hamster, dog, guinea pig, mouse, and chicken
and the designation "CYP1A2" includes the CYP1A2 protein from any
species known to have a CYP1A2 gene. In some embodiments, the
designation for a cytochrome p450 isozyme is the human isozyme.
[0086] In one embodiment, CYP1A2 is human CYP1A2 (Entrez Gene ID:
1544; reference protein sequence Genbank NP.sub.--000752), and
includes any allelic variants. Specifically, CYP1A2 includes any
allelic variants included in the list of human CYP1A2 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *16 alleles. Additional reference amino acid sequences for
human CYP1A2 include Genbank AAK25728, AAY26399, AAA35738,
AAA52163, AAA52163, AAF13599, AAH67424, AAH67425, AAH67426,
AAH67427, AAH67428, AAH67429, AAA52154, AAA52146, CAA77335, P05177,
Q6NWU3, Q6NWU5, Q9BXX7, and Q9UK49.
[0087] In one embodiment, CYP2A6 is human CYP2A6 (Entrez Gene ID:
1548; reference protein sequence Genbank NP.sub.--000753), and
includes any CYP2A6 allelic variants. Specifically, CYP2A6 includes
any allelic variants included in the list of human CYP2A6 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *22 alleles. Additional reference amino acid sequences for
human CYP2A6 include Genbank AAG45229, AAB40518, AAF13600,
AAH96253, AAH96254, AAH96255, AAH96256, AAA52067, CAA32097,
CAA32117, P11509, Q13120, and Q4VAU0.
[0088] In one embodiment, CYP2B6 is human CYP2B6 (Entrez Gene ID:
1555; reference protein sequence Genbank NP.sub.--000758), and
includes any CYP2B6 allelic variants. Specifically, CYP2B6 includes
any allelic variants included in the list of human CYP2B6 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *25 alleles. Additional reference amino acid sequences for
human CYP2B6 include Genbank AAF32444, AAD25924, ABB84469,
AAF13602, AAH67430, AAH67431, AAA52144, P20813, Q6NWU1, Q6NWU2, and
Q9UNX8.
[0089] In one embodiment, CYP2C8 is human CYP2C8 (Entrez Gene ID:
1558; reference protein sequence Genbank NP.sub.--110518), and
includes any CYP2C8 allelic variants. Specifically, CYP2B8 includes
any allelic variants included in the list of human CYP2C8 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *10 alleles. Additional reference amino acid sequences for
human CYP2C8 include Genbank CAH71307, AAR89907, CAA38578,
AAH20596, AAA35739, AAA35740, AAA52160, AAA52161, CAA35915,
CAA68550, P10632, Q5VX93, Q8WWB1, and Q9UCZ9.
[0090] In one embodiment, CYP2C9 is human CYP2C9 (Entrez Gene ID:
1559; reference protein sequence Genbank NP.sub.--000762), and
includes any CYP2C9 allelic variants. Specifically, CYP2C9 includes
any allelic variants included in the list of human CYP2C9 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *24 alleles. Additional reference amino acid sequences for
human CYP2C9 include Genbank CAH71303, AAP88931, AAT94065,
AAW83816, AAD13466, AAD13467, AAH20754, AAH70317, BAA00123,
AAA52159, AAB23864, P11712, Q5EDC5, Q5VX92, Q61RV8, Q8WW80, Q9UEH3,
and Q9UQ59.
[0091] In one embodiment, CYP2C19 is human CYP2C19 (Entrez Gene ID:
1557; reference protein sequence Genbank NP.sub.--000760), and
includes any CYP2C19 allelic variants. Specifically, CYP2C19
includes any allelic variants included in the list of human CYP2C19
allelic variants maintained by the Human Cytochrome P450 (CYP)
Allele Nomenclature Committee; more specifically it includes any of
the *1 through *21 alleles. Additional reference amino acid
sequences for human CYP2C19 include Genbank BAD02827, CAH73444,
CAH74068, AAV41877, AAL31347, AAL31348, AAA36660, AAB59426,
CAA46778, P33261, Q16743, Q767A3, Q8WZB1, and Q8WZB2.
[0092] In one embodiment, CYP2D6 is human CYP2D6 (Entrez Gene ID:
1565; reference protein sequence Genbank NP.sub.--000097), and
includes any CYP2D6 allelic variants. Specifically, CYP2D6 includes
any allelic variants included in the list of human CYP2D6 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *58 alleles. Additional reference amino acid sequences for
human CYP2D6 include Genbank AAS55001, ABB01370, ABB01371,
ABB01372, ABB01373, AAA35737, AAA53500, BAD92729, AAU87043,
AAH66877, AAH67432, AAH75023, AAH75024, AAI06758, AAI06759,
CAG30316, AAA52153, AAA36403, CAA30807, and P10635.
[0093] In one embodiment, CYP2E1 is human CYP2E1 (Entrez Gene ID:
1571; reference protein sequence Genbank NP.sub.--000764), and
includes any CYP2E1 allelic variants. Specifically, CYP2E1 includes
any allelic variants included in the list of human CYP2E1 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *7 alleles. Additional reference amino acid sequences for
human CYP2E1 include Genbank CAH70047, BAA00902, BAA08796,
AAA52155, AAD13753, AAF13601, CAI47002, AAH67433, AAH67435,
AAZ77710, AAA35743, AAD14267, P05181, Q16868, Q5VZD5, Q6LER5,
Q6NWT7, and Q6NWT9.
[0094] In one embodiment, CYP3A4 is human CYP3A4 (Entrez Gene ID:
1576; reference protein sequence Genbank NP.sub.--059488), and
includes any CYP3A4 allelic variants. Specifically, CYP3A4 includes
any allelic variants included in the list of human CYP3A4 allelic
variants maintained by the Human Cytochrome P450 (CYP) Allele
Nomenclature Committee; more specifically it includes any of the *1
through *20 alleles. Additional reference amino acid sequences for
human CYP3A4 include Genbank AAF21034, AAG32290, AAG53948,
EAL23866, AAF13598, CAD91343, CAD91645, CAD91345, AAH69418,
AAI01632, BAA00001, AAA35747, AAA35742, AAA35744, AAA35745,
CAA30944, PO.sub.5184, P08684, Q6GRK0, Q7Z448, Q86SK2, Q86SK3, and
Q9BZM0.
[0095] Various laboratory methods are known, including ones that
are commercially available, for detecting the presence of allelic
variants of cytochrome p450 isozymes in an individual or
determining the metabolizer phenotype of an individual for a
particular cytochrome p450 isozyme. Any suitable method known in
the art may be used. Methods include analyzing a blood sample from
the individual to determine the allelic variant of a particular
cytochrome p450 isozyme gene present in the individual (for example
by genotyping or haplotyping DNA or RNA from the gene using mass
spectrometry, gel electrophoresis, or TAQMAN assays; or analyzing
the protein sequence expressed by the gene). The metabolizer
phenotype of the individual can be inferred based on the known
properties of the allelic variants determined to be present in the
individual. Alternatively, the blood sample can be used to measure
enzyme activity of the cytochrome p450 isozyme using a suitable
assay and isozyme-selective substrate. Among suitable
isozyme-selective substrates are those used in the studies herein,
or those suggested in publications of the United States Food and
Drug Administration (FDA) directed to collecting cytochrome p450
isozyme data for regulatory submissions relating to an active
agent, for example, the document "Drug Interaction Studies-Study
Design, Data Analysis, and Implications For Dosing and Labeling;
Draft Guidance", dated September 2006, and available from the
Center for Drug Evaluation and Research (CDER) Guidance Documents
web page of the FDA website.
[0096] The ability of colchicine to affect enzyme activity of
various cytochrome P450 isozymes was determined in studies
described in the Examples.
[0097] In the inhibition study in which enzyme activity was
determined in human liver microsomes with the simultaneous presence
of colchicine and a cytochrome P450-isozyme specific substrate,
colchicine inhibited CYP2A6 and CYP2C8 at a statistically
significant level and activated CYP3A4 at a statistically
significant level. In these experiments, colchicine was not found
to affect enzyme activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, and CYP2E1 at a statistically significant
level.
[0098] In the induction study in which enzyme activity was
determined after preincubation of colchicine in the growth medium
of primary cultured human hepatocytes for 48 hours, CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 enzyme
activities were not induced by colchicine. Instead, colchicine was
determined to suppress enzymatic activity of each of the cytochrome
P450 isozymes examined, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, and CYP3A4, at a statistically significant
level.
[0099] Experiments were performed to look in more detail at the
suppression of CYP1A2 activity by colchicine observed in the
induction study. Effects of colchicine on enzyme activity levels
and mRNA expression levels of CYP1A2 were determined and compared
to the effects observed using vinblastine, another
microtubule-binding active agent. Colchicine was observed to
suppress enzyme activity by down-regulating mRNA expression,
whereas vinblastine suppressed neither enzyme activity nor mRNA
expression of CYP1A2.
[0100] Additionally, experiments were performed to identify
cytochrome P450 isozymes that metabolize colchicine. CYP1A2,
CYP2D6, and CYP2E1 showed no metabolism of colchicine. Metabolism
of colchicine by CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and
CYP3A4 was observed. The cytochrome P450 isozyme showing the
greatest amount of colchicine metabolism in these experiments was
CYP3A4.
[0101] The invention provides methods of using colchicine. These
methods include using colchicine in the treatment or prevention of
various diseases or conditions in a patient, including for example,
gout, attacks of acute gouty arthritis, pain in attacks of acute
gouty arthritis; a cystic disease (for example polycystic kidney
disease or cystic fibrosis), a lentiviral infection, demyelinating
diseases of central or peripheral origin, multiple sclerosis,
cancer, an inflammatory disorder such as rheumatoid arthritis,
glaucoma, Dupuytren's contracture, idiopathic pulmonary fibrosis,
primary amyloidosis, recurrent pericarditis, acute pericarditis,
asthma, postpericardiotomy syndrome, proliferative
vitreoretinopathy, Behcet's disease, Familial Mediterranean fever,
idiopathic thrombocytopenic purpura, primary biliary cirrhosis, and
pyoderma gangrenosum, or in enhancing bone formation or bone
mineral density. Using colchicine in the treatment or prevention of
a disease or condition in a patient can include administering
colchicine to a patient, dispensing colchicine to a patient, or
dispensing colchicine to a medical care worker for administering to
a patient.
[0102] In an embodiment, the method comprises informing a user that
colchicine affects the activity of a cytochrome P450 isozyme. In
one embodiment, the method comprises informing a user that
colchicine is metabolized by cytochrome P450 2A6, 2B6, 2C8, 2C9 or
2C19; inhibited cytochrome P450 2A6 or 2C8 enzyme activity in an in
vitro inhibition study; activated CYP3A4 enzyme activity in an in
vitro inhibition study; suppressed enzyme activity of cytochrome
P450 1A2, 2A6, 2C19, 2D6, or 2E1 in an in vitro induction study; or
suppressed mRNA expression of cytochrome P450 1A2 in an in vitro
induction study. In certain embodiments the cytochrome P450 isozyme
is a human enzyme. The method can further comprise providing the
user with colchicine.
[0103] Informing the user that colchicine affects the activity of a
cytochrome P450 isozyme includes providing a user with information
about any effect of colchicine on the activity of the cytochrome
P450 isozyme disclosed herein. Informing the user that colchicine
affects the activity of a cytochrome P450 isozyme includes
informing a user of any of the following: that colchicine is
metabolized by cytochrome P450 2A6, 2B6, 2C8, 2C9 or 2C19; that
colchicine inhibited cytochrome P450 2A6 or 2C8 enzyme activity in
an in vitro inhibition study; that colchicine activated CYP3A4
enzyme activity in an in vitro inhibition study; that colchicine
reduced enzyme activity of cytochrome P450 1A2, 2A6, 2B6, 2C8, 2C9,
2C19, 2D6, 2E1, or 3A4 in an in vitro induction study; that
colchicine significantly reduced enzyme activity of cytochrome P450
1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, or 3A4 in an in vitro induction
study, wherein a significant reduction is at least a 50% reduction;
that colchicine reduced mRNA expression of cytochrome P450 1A2 in
an in vitro induction study.
[0104] The method can further comprise informing the user that
administration of colchicine with a substance can affect the plasma
concentration, bioavailability, safety, efficacy, or a combination
comprising at least one of the foregoing of colchicine or the
substance. In some embodiments, the method further comprises
providing the user with the substance.
[0105] Informing the user that administration of colchicine with a
substance can affect the plasma concentration, bioavailability,
safety, efficacy, or a combination comprising at least one of the
foregoing of colchicine or the substance includes providing a user
with information about any effect of colchicine on plasma
concentration, bioavailability, safety, efficacy, or a combination
comprising at least one of the foregoing of colchicine or the
substance. This includes informing a user of any of the following:
that taking colchicine with an active agent can affect the
bioavailability, safety, or efficacy of the active agent or
colchicine; that administration of colchicine and a substance that
is a substrate, inhibitor, activator, inducer, or suppressor of
cytochrome P450 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, or 3A4 can
affect plasma concentration, bioavailability, safety, efficacy, or
a combination comprising at least one of the foregoing of
colchicine or the substance; that administration of colchicine and
a substance that is a known substrate, inhibitor, activator,
inducer, or suppressor of cytochrome P450 1A2, 2A6, 2B6, 2C8, 2C9,
2C19, 2D6, 2E1, or 3A4 can result in altered metabolism of
colchicine or the substance; that administration of colchicine with
a substance that is a known substrate of cytochrome P450 2A6 or 2C8
can result in reduced metabolism of the substance or increased
plasma concentration of the substance; that administration of
colchicine with a substance that is metabolized by CYP3A4 can
result in increased metabolism of the substance or decreased plasma
concentration of the substance; that administration of colchicine
with a substance that is metabolized by cytochrome P450 1A2, 2A6,
2B6, 2C8, 2C9, 2C19, 2D6, 2E1, or 3A4 can result in decreased
metabolism of the substance or increased plasma concentration of
the substance; that administration of colchicine with a substance
that is a known substrate of cytochrome P450 1A2 can result in
reduced metabolism of the substance or increased plasma
concentration of the substance; that administration of colchicine
with a substance that is metabolized by cytochrome P450 1A2, 2A6,
2B6, 2C8, 2C9, 2C19, 2D6, 2E1, or 3A4 can affect plasma
concentration, bioavailability, safety, efficacy, or a combination
comprising at least one of the foregoing of the substance or
colchicine; that administration of colchicine with a substance that
is a substrate or an inhibitor of cytochrome P450 2A6, 2B6, 2C8,
2C9, 2C19, or 3A4 can result in reduced metabolism of colchicine or
increased plasma concentration of colchicine; that caution is
recommended when administering colchicine with a substance, wherein
the substance is an active agent that has a sensitive plasma
concentration profile or a narrow therapeutic index; that there is
a potential active agent interaction between colchicine and a
substance that is a substrate of cytochrome P450 1A2, 2A6, 2B6,
2C8, 2C9, 2C19, 2D6, 2E1, or 3A4; that there is a potential active
agent interaction between colchicine and a substance that is an
inhibitor, activator, suppressor, or inducer of cytochrome P450
2A6, 2B6, 2C8, 2C9, or 2C19; that there is a potential active agent
interaction between colchicine and a substance that is a substrate
of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1; that caution is
recommended when colchicine and a substrate of CYP2A6, CYP2B6,
CYP2C9, 2C19, or 2D6 are administered to a patient having a poor
metabolizer phenotype for or reduced activity of the cytochrome
P450 isozyme; that the allelic variants of CYP2A6, CYP2B6, CYP2C9,
2C19, or 2D6 present in the patient can further affect a potential
active agent interaction between colchicine and a substance; or
that there is a potential active agent interaction of colchicine
with theophylline, warfarin, or phenyloin.
[0106] The effect of administration of colchicine with the
substance can be determined by comparison of the plasma
concentration, bioavailability, safety, efficacy, or a combination
comprising at least one of the foregoing of the substance with and
without administration of colchicine or by comparison of the plasma
concentration, bioavailability, safety, efficacy, or a combination
comprising at least one of the foregoing of colchicine with and
without administration of the substance.
[0107] In some embodiments, the method of using colchicine can
further comprise administering colchicine or a substance.
Administration may be to a patient by the patient, a medical care
worker, or other user. Colchicine can be administered in a
therapeutically effective amount. The substance can be an active
agent. The active agent can have a sensitive plasma concentration
profile or a narrow therapeutic index. The method can also comprise
monitoring a patient, for example, monitoring the patient for an
adverse reaction, a side effect, a subtherapeutic outcome, or a
symptom of an active agent interaction or monitoring a patient's
plasma concentration of colchicine or the substance. The method can
also comprise adjusting administration or dosing of the substance
or colchicine for the patient based on the results of monitoring,
for example, a determined plasma concentration of the active agent
or colchicine.
[0108] In all of the embodiments herein, a medical care worker can
determine the plasma concentration of a substance such as an active
agent, including colchicine, by performing or ordering the
performance of any suitable method. For example, the medical care
worker could order a test using blood drawn from the patient for
determining the plasma concentration of colchicine or the
substance.
[0109] Medical information provided in any of the methods described
herein concerning the effects of administering colchicine with an
additional substance may alternatively be provided in layman's
terms, so as to be better understood by patients or non-medical
professionals. Those of skill in the medical art are familiar with
the various layman's terms that can be used to describe the effects
of active agent interactions.
[0110] In an embodiment, the method of using colchicine comprises
obtaining colchicine from a container associated with published
material providing information that colchicine affects the activity
of a cytochrome P450 isozyme. Information can also be provided that
administering colchicine with a substance can affect plasma
concentration, bioavailability, safety, efficacy, or a combination
comprising at least one of the foregoing of the substance or
colchicine. The information provided by the published material can
comprise any combination of any information disclosed herein
concerning the effects of colchicine on the activity or expression
of a cytochrome P450 isozyme or any information disclosed herein
concerning the effects of colchicine when administered with a
substance on the plasma concentration, bioavailability, safety,
efficacy, or a combination comprising at least one of the foregoing
of the substance or colchicine. The method can also comprise
providing colchicine in the container providing such information.
The method can further comprise ingesting the colchicine or the
substance.
[0111] In an embodiment, the method comprises determining for a
patient to whom colchicine is going to be administered or is being
administered whether a substance that is currently being or will be
administered to the patient is a substrate of CYP1A2; and
determining risk for the patient of an adverse event during
coadministration of colchicine and the substance resulting from
reduced metabolism of the substance by CYP1A2.
[0112] Depending on the determined risk of an adverse event, such
as an active agent-related toxicity or a subtherapeutic outcome,
the methods can further comprise administering colchicine or the
substance to the patient. For example, if there is no risk of an
adverse event, or if the risk is determined to be acceptable,
colchicine and the substance can be administered to the patient.
Alternatively, if there is a risk of an adverse event, or if the
risk is determined to be unacceptable, either colchicine can be
administered to the patient but not the substance, or the substance
can be administered to the patient but not colchicine.
[0113] The method can further comprise determining that the patient
has a poor metabolizer phenotype for CYP2A6, CYP2C19, or CYP2D6 or
determining that the patient belongs to an ethnic group in which
there is a high frequency of a poor metabolizer phenotype of
CYP2A6, CYP2C19, or CYP2D6, e.g., for CYP2C19, an Asiatic or
Oceanic ethnic group.
[0114] Determining risk of an adverse reaction, such as a toxicity
or a subtherapeutic outcome, resulting from coadministration of
colchicine and a substance is based on an appropriate set of risk
parameters. As will be evident to those of skill in the art, the
risk parameters to be considered will be based upon factors which
influence the risk that a known or suspected adverse reaction will
occur if the patient receives colchicine with or without the
substance, and will vary depending upon the substance in question
for coadministration with colchicine. Factors that may define the
relevant risk parameters include effect of the substance or
colchicine on activity of the relevant cytochrome P450 isozyme(s),
e.g. CY3A4 or CYP1A2; the likelihood that certain preexisting
conditions may exist in the patient; information collected from the
patient including information relating to the patient's conduct;
the patient's past or ongoing medical treatment, such as other
procedures or medication which the patient may have received or is
still receiving; results of certain diagnostic tests which have
been performed; and the like. For example, if the substance is
theophylline, risk factors identified as reducing theophylline
clearance include the age of the patient, whether or not the
patient is a smoker, and whether the patient has any of the
following concurrent diseases or conditions: acute pulmonary edema,
congestive heart failure, cor-pulmonale, fever, hypothyroidism,
liver disease (e.g., cirrhosis or acute hepatitis), sepsis with
multi-organ failure, and shock. Information collected from the
patient for determining risk may be obtained prior to the initial
dispensation of colchicine or the substance to the patient or may
be obtained from the patient on a periodic basis. For example,
after treatment with colchicine and the substance is begun,
information on the onset of certain symptoms which may be
indicative of the need for changes in the patient's treatment
regimen may be obtained from the patient on a periodic basis. For
example if colchicine and theophylline are coadministered,
information on development of nausea or vomiting, particularly
repetitive vomiting, or other signs or symptoms consistent with
theophylline toxicity should be obtained.
[0115] Determining risk can comprise accessing a computer-hosted
database to obtain information relevant to assessing risk, for
example adverse reactions associated with an active agent, active
agent interactions, risk factors for an adverse reaction in
administration of an active agent, dosing, and the like. The
database may be in the form of a look-up table, or similar
structure, that provides output information based on the input of
information. The database can also be a component of a pharmacy
management system.
[0116] Pharmacy management systems are computer-based systems that
are widely used by commercial pharmacies to manage prescriptions
and to provide pharmacy and medical personnel with warnings and
guidance regarding active agents being administered to individuals.
Such systems typically provide alerts warning either or both of
medical care providers and patients when an active agent that may
be harmful to the particular patient is prescribed. For example,
such systems can provide alerts warning that a patient has an
allergy to a prescribed active agent, or is receiving concomitant
administration of an active agent that can have a dangerous
interaction with a prescribed active agent. U.S. Pat. Nos.
5,758,095, 5,833,599, 5,845,255, 6,014,631, 6,067,524, 6,112,182,
6,317,719, 6,356,873, and 7,072,840, each of which is incorporated
herein by reference, disclose various pharmacy management systems
and aspects thereof. Many pharmacy management systems are now
commercially available, e.g., CENTRICITY Pharmacy from BDM
Information Systems Ltd., General Electric Healthcare, Waukesha,
Wis., Rx30 Pharmacy Systems from Transaction Data Systems, Inc.,
Ocoee, Fla., SPEED SCRIPT from Digital Simplistics, Inc., Lenexa,
Kans., and various pharmacy management systems from OPUS-ISM,
Hauppauge, N.Y.
[0117] Alternatively, determining risk can comprise obtaining
information relevant to assessing risk from standard treatment
guidelines, textbooks, compendial literature, journals, drug
manufacturer guidelines, internet websites providing information on
active agent interactions (e.g., "Drug Interaction Checker" at the
MEDScape website or the drug interaction website maintained by Dr.
D. Flockhart, Indiana University School of Medicine); or FDA
requirements for particular active agents.
[0118] Diagnostic tests may be probative of the concentration of
one or more active agents, including a prescribed active agent, to
assure that appropriate dosing is maintained in the patient. Such
diagnostic testing may be conducted on any bodily fluid or waste
product of the patient, including the blood, serum, plasma, saliva,
semen or urine, as well as the feces. Diagnostic testing may also
be performed on a biopsy of any tissue of the patient or may
include genetic testing, which may be indicative of a genetic
predisposition to a particular adverse side effect. Other forms of
diagnostic testing, such as diagnostic imaging, or tests which may
be probative of the proper functioning of any tissue, organ, or
system are also contemplated. Preferably, appropriate information
or diagnostic test results are obtained and considered in
determining risk.
[0119] In an embodiment, the method comprises administering
colchicine to a patient; and monitoring the patient during
administration of colchicine if the patient is taking a substance
that is a known substrate of cytochrome P450 1A2, 2A6, 2C19, 2D6,
or 2E1. Adjusting administration of colchicine or the substance to
the patient to avoid an adverse event in the patient can be
performed.
[0120] In an embodiment, the method comprises determining that a
substance that is a substrate of cytochrome P450 1A2, 2A6, 2C19,
2D6, or 2E1 is administered to the patient; and adjusting
administration of colchicine or the substance to the patient to
avoid an adverse reaction.
[0121] In an embodiment, the method comprises determining that
colchicine reduced enzyme activity of cytochrome P450 1A2, 2A6,
2C19, 2D6, or 2E1 in an in vitro induction study; or reduced mRNA
expression of cytochrome P450 1A2 in an in vitro induction study;
and monitoring the patient during administration of colchicine if a
substance that is a substrate of cytochrome P450 1A2, 2A6, 2C19,
2D6, or 2E1 is coadministered to the patient. The method can
further comprise determining that a substance that is a substrate
of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 is coadministered to
the patient or adjusting administration of colchicine or the
substance to the patient to avoid an adverse reaction.
[0122] Such methods can include informing the patient receiving a
substance or the patient's medical care worker that administration
of colchicine with a substance can affect the plasma concentration,
bioavailability, safety, efficacy, or a combination comprising at
least one of the foregoing of colchicine or the substance. The
method can include informing the patient receiving a substance or
the patient's medical care worker of any information disclosed
herein about the effects of colchicine on cytochrome P450s and any
information disclosed herein about the effect of colchicine or the
substance on the plasma concentration, bioavailability, safety,
efficacy, or a combination comprising at least one of the foregoing
of colchicine or the substance when colchicine is used with the
substance.
[0123] Determining that a substance that is a known substrate,
inhibitor, or inducer of a particular cytochrome P450 isozyme is
administered to a patient in need of colchicine therapy can be
performed by consulting with the patient regarding substances,
e.g., medications, taken in by the patient; a medical care worker
administering medications to the patient; a prescription database
including medications prescribed to the patient; or by any other
method known in the art.
[0124] Determining that colchicine is metabolized by cytochrome
P450 2A6, 2B6, 2C8, 2C9 or 2C19; inhibited cytochrome P450 2A6 or
2C8 enzyme activity in an in vitro inhibition study; activated
CYP3A4 enzyme activity in an in vitro inhibition study; suppressed
enzyme activity of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 in
an in vitro induction study; or suppressed mRNA expression of
cytochrome P450 1A2 in an in vitro induction study or determining
that co-administration of colchicine and a substance that is a
substrate of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 may result
in an increased plasma concentration of the substance, or that
co-administration of colchicine and a substance that is a substrate
of cytochrome P450 1A2, 2A6, 2C19, 2D6, or 2E1 may result in
decreased metabolism of the substance can be performed by
consulting the package insert for the colchicine product or for the
substance administered to the patient; consulting a database
including prescribing information and potential risks for
colchicine or the substance, e.g., a pharmacy management system; or
by any other method known in the art.
[0125] Monitoring the patient can comprise monitoring the patient's
plasma concentration of colchicine or the substance; monitoring the
patient for symptoms of an active agent interaction between the
substance and colchicine; monitoring the patient for an adverse
reaction (e.g., a toxicity or a subtherapeutic outcome) resulting
from administration of the substance and colchicine; monitoring the
patient for an adverse reaction (e.g., a toxicity or a
subtherapeutic outcome) associated with colchicine; monitoring the
patient for decreased efficacy of colchicine; monitoring the
patient for an adverse reaction associated with elevated plasma
concentration of the substance; monitoring the patient for an
adverse reaction or side effect associated with the substance;
monitoring the patient for a substance-associated toxicity; or
monitoring the patient for a symptom of elevated plasma
concentration of the substance.
[0126] Monitoring the patient can be monitoring any appropriate
patient-specific, disease-specific, or substance-specific parameter
appropriate to avoid or safely manage an active agent interaction.
Monitoring the patient can be, for example, monitoring the patient
for an adverse reaction, a subtherapeutic outcome, a side effect,
or a symptom of an active agent interaction for example by physical
examination or visual identification; monitoring the blood level of
colchicine or the substance in the patient; monitoring clinical
laboratory tests appropriate for colchicine, the substance, or a
medical diagnosis for the patient; monitoring therapeutic effect of
colchicine or the substance on the patient's condition; monitoring
occurrence in the patient of a known side effect or adverse
reaction of colchicine or the substance; monitoring the patient for
a symptom of an active agent interaction between the substance and
colchicine; monitoring the patient for an adverse reaction or side
effect associated with altered plasma concentration of colchicine
or the substance; monitoring the patient for occurrence of an
unexpected response during treatment; monitoring changes in
control, signs, or symptoms of a condition of the patient, or
determining a complete list of medical diagnoses for the patient.
Monitoring the patient can be performed by the patient or by a
medical care worker.
[0127] Most active agents have adverse side effects having widely
variable incidence, according to individual sensitivity. For
colchicine, the most frequently reported adverse reactions to
colchicine therapy are abdominal pain with cramps, diarrhea,
nausea, and vomiting. Less frequently or rarely reported adverse
reactions associated with colchicine therapy include anorexia,
agranulocytosis, allergic dermatitis, allergic reactions, alopecia,
angioedema, aplastic anemia, bone marrow depression, myopathy,
neuropathy, skin rash, thrombocytopenic disorder, and
urticaria.
[0128] Determining that a patient experiences an adverse reaction
can be performed by obtaining information from the patient
regarding onset of certain symptoms which may be indicative of the
adverse reaction, results of diagnostic tests indicative of the
adverse reaction, and the like.
[0129] Determining the level of metabolism of a substance or
colchicine in a subject may be performed for example by determining
plasma concentrations of colchicine or the substance or of an
appropriate metabolite of colchicine or the substance, or any other
methods known in the art.
[0130] Adjusting administration of colchicine or the substance to
the patient to avoid an adverse reaction or a subtherapeutic
outcome, or adjusting dosing regimens can be performed by one of
ordinary skill in the art, taking into consideration the physiology
of the patient, including such factors as the age, sex, and health
of the patient, as well as active agents the patient may be taking
at the time. Optionally, the patient can be monitored at the
initial, or a subsequent, stage of treatment to ensure therapeutic
plasma levels of colchicine or the substance are achieved or
maintained.
[0131] In another aspect, herein disclosed are methods for using
colchicine which methods involve the use of pharmacy management
systems.
[0132] In one aspect, one such method comprises a pharmacy
receiving a prescription for colchicine for a patient who is
suffering from a condition treatable with colchicine and who is
concomitantly being treated with a second active agent that is a
substrate of CYP1A2, followed by the pharmacy dispensing colchicine
in response to receipt of the prescription, wherein the dispensing
is preceded by entry into a first computer readable storage medium,
in functional communication with a computer, of a unique patient
identifier for said patient and at least one active agent
identifier for colchicine linked to the patient identifier so as to
indicate that colchicine is to be administered to the patient. The
computer is programmed to issue an active agent interaction alert
when the at least one active agent identifier for colchicine is
entered linked to the patient identifier so as to indicate that
colchicine is to be administered to the patient and when the
patient identifier is also linked to an identifier indicating that
a second active agent that is a substrate of CYP1A2 is being
concomitantly administered to the patient. Upon entry of the at
least one active agent identifier for colchicine linked to the
patient identifier, an active agent interaction alert is issued to
one or more of a pharmacy technician, a pharmacist, or a pharmacy
customer obtaining the colchicine, said alert indicating that a
second active agent that is a substrate of CYP1A2 is being
concomitantly administered to the patient and that prior to the
colchicine being dispensed, the prescribed colchicine and second
active agent dosing regimens must be reviewed and, if necessary
adjusted by the prescribing medical care worker.
[0133] The active agent interaction alert is preferably issued as
one or both of a written warning on a display screen of the
pharmacy management computer system, and a printed warning. The
printed warning may be attached to or packaged with the dispensed
prescription.
[0134] Methods of using colchicine include methods in which the
user is a patient in need of treatment with colchicine and
additionally comprising administering colchicine and an active
agent to the patient. The patient in need of treatment with
colchicine may be, for example, a human patient, a patient in need
of treatment of attacks of acute gouty arthritis and pain in
attacks of acute gouty arthritis, a cystic disease, for example
polycystic kidney disease or cystic fibrosis, a lentiviral
infection, demyelinating diseases of central or peripheral origin,
multiple sclerosis, cancer, an inflammatory disorder such as
rheumatoid arthritis, glaucoma, Dupuytren's contracture, idiopathic
pulmonary fibrosis, primary amyloidosis, recurrent pericarditis,
acute pericarditis, asthma, postpericardiotomy syndrome,
proliferative vitreoretinopathy, Behcet's disease, Familial
Mediterranean fever, idiopathic thrombocytopenic purpura, primary
biliary cirrhosis, and pyoderma gangrenosum, or in enhancing bone
formation or bone mineral density, a patient receiving prophylactic
colchicine treatment, or a patient undergoing colchicine therapy.
The active agent administered to the patient with colchicine can be
for treatment or prophylaxis of a condition of the patient other
than the condition needing treatment with colchicine. The amount of
colchicine or the active agent administered may be a
therapeutically effective amount.
[0135] In an embodiment, a method can additionally include
monitoring the patient's plasma concentration of the active agent
or colchicine. When colchicine is administered together with
another active agent, methods of use can include determining the
plasma concentration of the active agent or colchicine and
adjusting the dosing regimen of the active agent or colchicine for
the patient based on the determined plasma concentration of the
active agent or colchicine.
[0136] When the substance administered with colchicine is an NTI or
sensitive plasma concentration profile active agent, methods using
a blood test to monitor plasma levels of the NTI or sensitive
plasma concentration profile active agent comprise administering to
a patient colchicine and the NTI or sensitive plasma concentration
profile active agent, and monitoring the blood levels of the NTI or
sensitive plasma concentration profile active agent. Methods can
also include adjusting dosing of the NTI or sensitive plasma
concentration profile active agent for the patient based on the
determined plasma concentration of the active agent.
[0137] In some embodiments, the NTI active agent comprises
warfarin. Warfarin, 3-(a-acetonylbenzyl)-4-hydroxycoumarin, is an
anticoagulant, which is eliminated by metabolism by cytochrome p450
isoforms including CYP2C9, CYP2C19, CYP2C8, CYP2C18, CYP1A2, and
CYP3A4. Warfarin has a narrow therapeutic index such that too
little can lead to excessive clotting, while excessive warfarin can
lead to excessive bleeding. The dosing of warfarin is
individualized according to the patient's sensitivity to the active
agent as indicated, for example, by the Prothrombin
Time/International Normalized Ratio (PT/INR). The PT/INR gives an
indication of how fast blood is clotting. The recommended initial
dose is 2-5 mg/day, with 2-10 mg/day as the maintenance dose.
Warfarin tablets for oral administration include tablets comprising
1, 2, 2.5, 3, 4, 5, 6, 7.5, and 10 mg of warfarin. The INR may be
adjusted to 2.0-4.5, or 2.0-3.0 or 2.5-3.5 depending on whether the
warfarin is being administered to treat venous thromboembolism,
non-valvular atrial fibrillation, post-myocardial infarction, heart
valve prophylaxis, or recurrent systemic embolism.
[0138] In the PT test, a reagent which induces coagulation is added
to a sample of the patient's plasma. The reagent typically
primarily comprises thromboplastin and calcium chloride. Many
commercially available PT reagents contain crude thromboplastin
extracted from natural sources, e.g., rabbit brain, rabbit
brain/lung mixtures, human placenta, or bovine brain, although
recombinant thromboplastin may also be employed. Prothrombin time
assays are performed by mixing the plasma sample and reagent at a
constant temperature such as 37.degree. C., and monitoring the
progress of the reaction until a perceptible clot (or "gel clot")
is detected. The development of a gel clot is the end point of the
reaction. This end point may be detected in various ways such as by
viscosity change, by electrode reaction, and, most commonly, by
photometric means. The test result is generally compared to a
result using a normal (control) plasma and converted to an INR.
[0139] The International Normalized Ratio, or INR, was developed to
standardize PT values, so that test results from different
thromboplastins and coagulation analyzers become equivalent. Under
the INR system, a thromboplastin is assigned an International
Sensitivity Index (ISI) value. The ISI indicates the relative
sensitivity of the thromboplastin compared to an international
reference thromboplastin. If a thromboplastin has the same
sensitivity as the reference thromboplastin, then its ISI is 1.0. A
higher ISI value indicates that a thromboplastin is less sensitive
than the reference thromboplastin. The ISI is used in the following
formula to calculate an INR value from a PT value: INR=(patient
PT/mean normal PT)ISI. The ISI is usually determined by the
thromboplastin manufacturer. Different ISI values are assigned for
different models or classes of coagulation analyzers.
[0140] In an embodiment of the method of using colchicine in which
the substance is warfarin, the method comprises administering to a
patient colchicine and warfarin, and monitoring the blood levels of
warfarin and colchicine or monitoring the Prothrombin
Time/International Normalized Ratio.
[0141] In another embodiment, the method comprises administering
colchicine and warfarin to a patient in need of colchicine and an
anticoagulant, and monitoring the Prothrombin Time/International
Normalized Ratio. Monitoring the Prothrombin Time/International
Normalized Ratio may be performed daily, every other day, weekly,
every other week, or monthly, for example. The method may further
comprise providing to the patient or medical care worker
instructions regarding measuring the Prothrombin Time/International
Normalized Ratio daily, every other day, weekly, every other week,
monthly, or according to another schedule or time criteria.
[0142] The NTI active agent can also comprise phenyloin. Phenyloin,
5,5-diphenylhydantoin, is an antiepileptic active agent useful in
the treatment of epilepsy which is eliminated by metabolism by
cytochrome P450 isoforms including CYP1A2, CYP2C9, CYP2C19, and
CYP3A4. Phenyloin has a narrow therapeutic index such that too
little can lead to insufficient results and excessive phenyloin can
lead to phenyloin toxicity. The typical clinically effective serum
level is about 10 to about 20 mg/mL. The recommended initial dose
is one 100 mg capsule 3 to 4 times per day, with 300 mg/day dose in
three divided doses or one single dose per day. The dosing of
phenyloin can be individualized according to the patient's
sensitivity to the active agent by measuring plasma concentration
of phenyloin.
[0143] In an embodiment of the method of using colchicine in which
the substance is phenyloin, the method comprises administering
colchicine and phenyloin to a patient in need of colchicine and an
antiepileptic, and monitoring the blood levels of phenyloin.
[0144] The NTI active agent can also comprise theophylline.
Theophylline is a bronchodilator structurally classified as a
xanthine derivative. Theophylline is a substrate of CYP1A2 and
CYP2E1. Adverse reactions associated with theophylline are
generally mild when peak serum theophylline concentrations are less
than about 20 .mu.g/mL and mainly consist of transient
caffeine-like adverse effects such as nausea, vomiting, headache,
and insomnia. When peak serum theophylline concentrations exceed 20
.mu.g/mL, however, theophylline produces a wide range of adverse
reactions including persistent vomiting, cardiac arrhythmias, and
intractable seizures which can be lethal. The dose of theophylline
must be individualized on the basis of peak serum theophylline
concentration measurements in order to achieve a dose that will
provide maximum potential benefit with minimal risk of adverse
events.
[0145] In an embodiment, the method of using colchicine when the
substance is theophylline comprises administering colchicine and
theophylline to a patient in need of colchicine and a
bronchodilator; monitoring the blood levels of theophylline; and
adjusting dosing of theophylline to avoid an adverse reaction.
[0146] Also disclosed herein are methods of manufacturing a
colchicine pharmaceutical product.
[0147] In one embodiment, the method comprises packaging a
colchicine dosage form with published material providing
information on the effects of colchicine on a cytochrome p450
isozyme. The information can include any information disclosed
herein concerning colchicine effects on a cytochrome p450 isozyme.
The information can also include any information disclosed herein
about the effects of administering colchicine and a substance on
the plasma concentration, bioavailability, safety, efficacy, or a
combination comprising at least one of the foregoing of colchicine
or the substance when the substance is used with colchicine.
[0148] The invention provides articles of manufacture.
[0149] In some embodiments, the article of manufacture comprises a
container containing a dosage form of colchicine and labeling or
published material, e.g., as a product insert or a patient package
insert. The published material can indicate quantities of the
components to be administered, guidelines for administration,
safety issues, and the like.
[0150] In some embodiments, the container is associated with
published material informing that colchicine affects a cytochrome
p450 isozyme. The information provided by the published material
can include any information disclosed herein concerning effects of
colchicine on a cytochrome p450 isozyme. The published material
comprised in the article of manufacture can also include any
information disclosed herein concerning the effect of administering
colchicine and a substance on the plasma concentration,
bioavailability, safety, efficacy, or a combination comprising at
least one of the foregoing of colchicine or the substance when the
substance is used with colchicine. The published material may be in
the form of printed labeling, or in some other form.
[0151] Also disclosed herein is an article of manufacture
comprising packaging material and a dosage form contained within
the packaging material, wherein the dosage form comprises
colchicine, and wherein the packaging material comprises a label
approved by a regulatory agency for the product. Examples of
regulatory agencies are the US FDA or the European Agency for the
Evaluation of Medicinal Products (EMEA). The label can inform of
any information disclosed herein about the effect of colchicine on
metabolism by a cytochrome p450 isozyme or any information
disclosed herein about the effects of administering colchicine and
a substance on the plasma concentration, bioavailability, safety,
efficacy, or a combination comprising at least one of the foregoing
of colchicine or the substance when the substance is used with
colchicine.
[0152] In embodiments of the articles of manufacture, the dosage
form will typically be contained in a suitable container capable of
holding and dispensing the dosage form and which will not
significantly interact with the active agent(s) in the dosage form.
Further, the container will be in physical relation with the
published material. The published material may be associated with
the container by any means that maintains physical proximity of the
two. By way of example, the container and the published material
can both be contained in a packaging material such as a box or
plastic shrink wrap. Alternatively, the published material can be
bonded to the container, such as with glue that does not obscure
the published material, or with other bonding or holding means. Yet
another alternative is that the published material is placed within
the container with the dosage form.
[0153] Someone can also hand the published material to the patient,
for example a pharmacist can hand a product insert, patient package
insert, or medication guide to a patient in conjunction with
dispensing the dosage form. The published material may be a product
insert, patient package insert, medication guide, flyer, brochure,
or a packaging material for the dosage form such as a bag, or the
like.
[0154] In any of the embodiments disclosed herein the published
material or information associated with or provided by a container
can be contained in any fixed and tangible medium. For example, the
information can be part of a leaflet, brochure, or other printed
material provided with a container or separate from a container.
The information can also take the form of a flyer, advertisement,
or the label for marketing the active agent approved by a
regulatory agency. The information can also be recorded on a
compact disk, DVD or any other recording or electronic medium.
[0155] The container can be in the form of bubble or blister pack
cards, optionally arranged in a desired order for a particular
dosing regimen. Suitable blister packs that can be arranged in a
variety of configurations to accommodate a particular dosing
regimen are well known in the art or easily ascertained by one of
ordinary skill in the art.
[0156] Colchicine dosage forms existing as liquids, solutions,
emulsions, or suspensions can be packaged in a container for
convenient dosing of pediatric or geriatric patients. For example,
prefilled droppers (such as eye droppers or the like), prefilled
syringes, and similar containers housing the liquid, solution,
emulsion, or suspension form are contemplated.
[0157] Colchicine can be formulated as a dosage form for
administration where the formulation generally contains colchicine
and a pharmaceutically acceptable excipient. As used herein,
"pharmaceutically acceptable excipient" means any other component
added to the pharmaceutical formulation other than the active
agent. Excipients may be added to facilitate manufacture, enhance
stability, control release, enhance product characteristics,
enhance bioavailability, enhance patient acceptability, etc.
Pharmaceutical excipients include carriers, fillers, binders,
disintegrants, lubricants, glidants, compression aids, colors,
sweeteners, preservatives, suspending agents, dispersing agents,
film formers, flavors, printing inks, buffer agents, pH adjusters,
preservatives etc.
[0158] The substance used with colchicine in the methods and
articles of manufactures described herein may have certain effects,
direct or indirect, on the activity of a cytochrome P450
enzyme.
[0159] In any of the above methods or articles, the substance can
be an active agent.
[0160] Examples of substrates of CYP1A2 include aminophylline,
amitriptyline, caffeine, clomipramine, clozapine, cyclobenzaprine,
estradiol, fluvoxamine, haloperidol, imipramine, mexiletine,
naproxen, olanzapine, ondansetron, phenacetin, acetaminophen,
propranolol, riluzole, ropivacaine, tacrine, theophylline,
tizanidine, verapamil, (R)-warfarin, zileuton, and zolmitriptan.
Examples of inhibitors of CYP1A2 include amiodarone, cimetidine, a
fluoroquinolone (e.g., ciprofloxacin, gatifloxacin, levofloxacin,
lomefloxacin, moxifloxacin, norfloxacin, or ofloxacin),
fluvoxamine, furafylline, interferon, methoxsalen, and mibefradil.
Examples of inducers of CYP1A2 include chemicals released from
digestion of broccoli, brussel sprouts, and char-grilled meat;
chemicals inhaled when smoking tobacco; insulin, methyl
cholanthrene, modafinil, nafcillin, beta-naphthoflavone, and
omeprazole.
[0161] Examples of substrates of CYP2A6 include aflatoxin B.sub.1,
cotinine, coumarin, 1,7-dimethylxanthine, disulfuram, fadrozole,
halothane, losigamone, letrozole, methoxyflurane, nicotine,
tobacco-specific nitrosamines, SM-12502, tegafur, and valproic
acid. Examples of inhibitors of CYP2A6 include tranylcypromine,
methoxsalen, pilocarpine, and tryptamine. Examples of inducers of
CYP2A6 include dexamethasone and pyrazole.
[0162] Examples of substrates of CYP2B6 include bupropion,
cyclophosphamide, efavirenz, ifosfamide, and methadone. Examples of
inhibitors of CYP2B6 include thiotepa and ticlopidine. Examples of
inducers of CYP2B6 include phenobarbital and rifampin.
[0163] Examples of substrates of CYP2C8 include amodiaquine,
cerivastatin, paclitaxel, repaglinide, and torsemide. Examples of
inhibitors of CYP2C8 include quercetin, a glitazone (e.g.,
rosiglitazone or pioglitazone), gemfibrozil, montelukast, and
trimethoprim. Examples of inducers of CYP2C8 include rifampin.
[0164] Examples of substrates of CYP2C9 include diclofenac,
ibuprofen, meloxicam, S-naproxen, piroxicam, suprofen, tolbutamide,
glipizide, losartan, irbesartan, glyburide (glibenclamide),
glipizide, glimepiride, amitriptyline, celecoxib, fluoxetine,
fluvastatin, nateglinide, phenyloin, rosiglitazone, tamoxifen,
torsemide, and S-warfarin. Examples of inhibitors of CYP2C9 include
amiodarone, fenofibrate, fluconazole, fluvastatin, fluvoxamine,
isoniazid, lovastatin, phenylbutazone, probenicid, sertraline,
sulfamethoxazole, sulfaphenazole, teniposide, voriconazole, and
zafirlukast. Examples of inducers of CYP2C9 include rifampin and
secobarbital.
[0165] Examples of substrates of CYP2C19 include the proton pump
inhibitors: lansoprazole, omeprazole, pantoprazole, and E-3810; the
anti-epileptics: diazepam, phenyloin, fosphenyloin, S-mephenyloin,
and phenobarbitone (Phenobarbital); as well as amitriptyline,
carisoprodol, citalopram, clomipramine, cyclophosphamide,
hexobarbital, imipramine, indomethacin, R-mephobarbital,
moclobemide, nelfinavir, nilutamide, primidone, progesterone,
proguanil, propranolol, teniposide, and R-warfarin. Examples of
inhibitors of CYP2C19 include chloramphenicol, cimetidine,
felbamate, fluoxetine, fluvoxamine, indomethacin, ketoconazole,
lansoprazole, modafinil, omeprazole, oxcarbazepine, probenicid,
ticlopidine, and topiramate. Examples of inducers of CYP2C19
include carbamazepine, norethindrone, prednisone, and nifampin
(rifampicin).
[0166] Examples of substrates of CYP2D6 include carvedilol,
S-metoprolol, propafenone, timolol; amitriptyline, clomipramine,
desipramine, imipramine, paroxetine; haloperidol, perphenazine,
risperidone, thioridazine; alprenolol, amphetamine, aripiprazole,
atomoxetine, bufuralol, chlorpheniramine, chlorpromazine, codeine,
debrisoquine, dexfenfluramine, dextromethorphan, duloxetine,
encamide, flecamide, fluoxetine, fluvoxamine, lidocaine,
metoclopramide, methoxyamphetamine, mexiletine, minaprine,
nebivolol, nortriptyline, ondansetron, perhexyline, phenacetin,
phenformin, propranolol, sparteine, tamoxifen, tramadol, and
venlafaxine. Examples of inhibitors of CYP2D6 include amiodarone,
bupropion, celecoxib, chlorpromazine, chlorpheniramine, cimetidine,
citalopram, clomipramine, cocaine, doxepin, doxorubicin,
duloxetine, escitalopram, fluoxetine, halofantrine,
red-haloperidol, levomepromazine, metoclopramide, methadone,
mibefradil, midodrine, moclobemide, paroxetine, quinidine,
ranitidine, ritonavir, sertraline, terbinafine, ticlopidine,
histamine H1 receptor antagonists, diphenhydramine,
chlorpheniramine, clemastine, perphenazine, hydroxyzine, and
tripelennamine. Examples of inducers of CYP2D6 include rifampicin
and dexamethasone.
[0167] Examples of substrates of CYP2E1 include enflurane,
halothane, isoflurane, methoxyflurane, sevoflurane; acetaminophen,
aniline, benzene, chlorzoxazone, ethanol, N,N-dimethyl formamide,
and theophylline. Examples of inhibitors of CYP2E1 include
diethyl-dithiocarbamate and disulfuram. Examples of inducers of
CYP2E1 include ethanol and isoniazid.
[0168] Examples of substrates of CYP3A4 include clarithromycin,
erythromycin, telithromycin: quinidine; alprazolam, diazepam,
midazolam, triazolam; cyclosporine, tacrolimus (FK506); indinavir,
nelfinavir, ritonavir, saquinavir; cisapride; astemizole,
chlorpheniramine, terfenadine; amlodipine, diltiazem, felodipine,
lercanidipine, nifedipine, nisoldipine, nitrendipine, verapamil;
atorvastatin, cerivastatin, lovastatin, simvastatin; estradiol,
hydrocortisone, progesterone, testosterone; alfentanyl,
aripiprazole, buspirone, cafergot, caffeine, cilostazol, cocaine,
codeine, dapsone, dextromethorphan, docetaxel, domperidone,
eplerenone, fentanyl, finasteride, gleevec, haloperidol,
irinotecan, Levo-Alpha Acetyl Methadol (LAAM), lidocaine,
methadone, nateglinide, odanestron, pimozide, propranolol, quinine,
salmeterol, sildenafil, sirolimus, tamoxifen, taxol, terfenadine,
trazodone, vincristine, zaleplon, and zolpidem. Examples of
inhibitors of CYP3A4 include HIV Antivirals (e.g., delavirdine,
indinavir, nelfinavir, and ritonavir); amiodarone, aprepitant,
cinchloramphenicol, cimetidine, clarithromycin,
diethyl-dithiocarbamate, diltiazem, erythromycin, fluconazole,
fluvoxamine, gestodene, grapefruit juice, Seville orange juice,
imatinib, itraconazole, ketoconazole, mifepristone, nefazodone,
norfloxacin, norfluoxetine, mibefradil, star fruit, verapamil, and
voriconazole. Examples of inducers of CYP3A4 include HIV Antivirals
(e.g., efavirenz, and nevirapine); barbiturates (e.g.,
allobarbital, amobarbital, aprobarbital, alphenal, barbital,
brallobarbital, mephobarbital, secobarbital, and phenobarbital),
carbamazepine, efavirenz, glucocorticoids (e.g., prednisone,
prednisilone, methylprednisilone, dexamethasone, and
hydrocortisone), modafinil, nevirapine, phenyloin, rifampin, St.
John's wort, troglitazone, oxcarbazepine, pioglitazone, and
rifabutin.
[0169] In any of the embodiments described herein, the substance
can be a sensitive plasma concentration profile active agent.
Examples of a sensitive plasma concentration profile active agent
include cyclophosphamide, efavirenz, fosphenyloin, glimepiride,
mexiletine, phenyloin, progesterone, tamoxifen, theophylline,
warfarin, and any active agent having a narrow therapeutic
index.
[0170] In any of the embodiments described herein, the substance
can be an active agent having a narrow therapeutic index. Examples
of narrow therapeutic index active agents include aprindine,
carbamazepine, clindamycin, clonazepam, clonidine, cyclosporine,
digitoxin, digoxin, disopyramide, ethinyl estradiol, ethosuximide,
fosphenyloin, guanethidine, isoprenaline, lithium, methotrexate,
phenobarbital, phenyloin, pimozide, prazosin, primidone,
procainamide, quinidine, sulfonylurea compounds (e.g.,
acetohexamide, glibenclamide, gliclazide, glyclopyramide,
tolazamide, tolbutamide), tacrolimus, theophylline compounds (e.g.,
aminophylline, choline theophylline, diprophylline, proxyphylline,
and theophylline), thioridazine, valproic acid, warfarin, and
zonisamide.
[0171] The invention is further illustrated by the following
examples.
Example 1
Colchicine Inhibition of Cytochrome P450 Isozymes in Human
Microsomes
[0172] The study of this example was performed to determine the
potential of colchicine to inhibit the activities of cytochrome
P450 isoforms CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes. Human liver
microsomes were incubated in the presence of colchicine and a
substrate selective for each CYP isoform. A table of the substrate,
substrate concentration, solvent, metabolite formed and metabolite
assay method for each CYP isozyme studied is below.
TABLE-US-00001 TABLE 1 Isoform-selective substrates for cytochrome
P450 isozymes. CYP Isoform-selective Substrate Metabolite isoform
substrate concentration Solvent Metabolite formed Assay CYP1A2
Phenacetin 50 .mu.M ACN acetaminophen LC/MS CYP2A6 Coumarin 8 .mu.M
ACN 7-hydroxycoumarin HPLC-UV CYP2B6 S-Mephenytoin 1 mM ACN
nirvanol LC/MS CYP2C8 Paclitaxel 5 .mu.M ACN 6-hydroxypaclitaxel
HPLC-UV CYP2C9 Tolbutamide 150 .mu.M ACN
4'-methylhydroxytolbutamide LC/MS CYP2C19 S-Mephenytoin 50 .mu.M
ACN 4'-hydroxymephenytoin LC/MS CYP2D6 Dextromethorphan 5 .mu.M
Water dextrorphan LC/MS CYP2E1 Chlorzoxazone 50 .mu.M ACN
6-hydroxychlorzoxazone LC/MS CYP3A4 Testosterone 100 .mu.M ACN
6.beta.-hydroxytestosterone HPLC-UV
[0173] Colchicine stock solutions were prepared in water at 100
times the final concentration and added to incubation mixtures to
obtain final concentrations of 0.2, 2, 10, 20, and 50 .mu.M, each
containing 1% water and 1% acetonitrile.
[0174] Microsomes were prepared by differential centrifugation of
liver homogenates pooled from at least ten human donors.
[0175] Incubation mixtures were prepared in 0.1 M Tris buffer and
contained microsomes (0.25 mg protein/mL for CYP2C9, CYP2D6,
CYP2E1, and CYP3A4; 0.5 mg protein/mL for CYP1A2, CYP2A6, CYP2B6,
CYP2C8, and CYP2C19), colchicine, and a CYP isoform-selective
substrate. All incubations were conducted at 37.+-.1.degree. C. in
a shaking water bath. After a 5 minute preincubation, NADPH
regenerating system (NRS) was added to initiate the reaction.
CYP2A6 and CYP3A4 incubations were for 10 minutes. All other
incubations were for 30 minutes.
[0176] Incubations for CYP2C8 were terminated by adding 1.5 mL of
ACN, while all other incubations were terminated by adding 1.0 mL
of methanol. Samples were transferred to cryovials and analyzed for
metabolite after storage at -70.degree. C. Three replicates were
performed at each concentration of colchicine for each cytochrome
P450 isozyme.
[0177] To verify that the test system was responsive to inhibitors,
a positive control using ketoconazole, a selective inhibitor of
CYP3A4, was added to a microsome incubation. Four replicates were
performed. The test system was considered responsive to inhibitors
since the mean specific activity of CYP3A4 in the positive control
samples treated with ketoconazole was <22.1% of the mean
specific activity in the corresponding vehicle control samples.
[0178] Vehicle control experiments were performed to establish a
baseline value for enzyme activity. Incubation mixtures without
added colchicine were prepared as described above. Reactions were
initiated, run, and terminated as described above. Four replicates
were performed.
[0179] Colchicine interference control samples were also included
to eliminate the possibility of interference by colchicine or its
metabolites in detection of the metabolite formed from the
isoform-selective substrate. Incubation mixtures were prepared as
described above containing 50 .mu.M colchicine, but no added
isoform-selective substrate. In place of the substrate, substrate
solvent was added to yield a final concentration of 1%. Reactions
were initiated, run, and terminated as described above. Two
replicates of the interference control experiments were performed.
No interference was detected in any of the metabolite assay methods
used.
[0180] Results for each CYP isoform, in the presence and absence of
colchicine, are reported in Tables 2-10.
TABLE-US-00002 TABLE 2 Colchicine Effects on CYP1A2 Activity in
Pooled Human Liver Microsomes Acetaminophen formation Specific
Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg protein)
Percent (.mu.M) (.mu.M) Individual Mean .+-.SD Individual Mean
.+-.SD of VC 0 0.21210 0.212 0.209 .+-.0.0105 28.3 27.8 .+-.1.40
100 (VC) 0.21693 0.217 28.9 0.19336 0.193 25.8 0.21273 0.213 28.4
0.2 0.18336 0.183 0.190 .+-.0.00549 24.4 25.3 .+-.0.732 90.9
0.19283 0.193 25.7 0.19291 0.193 25.7 2 0.20043 0.200 0.208
.+-.0.00942 26.7 27.7 .+-.1.26 99.5 0.20457 0.205 27.3 0.21842
0.218 29.1 10 0.21659 0.217 0.205 .+-.0.0106 28.9 27.3 .+-.1.42
98.0 0.19632 0.196 26.2 0.20098 0.201 26.8 20 0.18961 0.190 0.188
.+-.0.00232 25.3 25.1 .+-.0.309 90.0 0.18534 0.185 24.7 0.18905
0.189 25.2 50 0.19763 0.198 0.198 .+-.0.00349 26.4 26.4 .+-.0.465
94.7 0.20139 0.201 26.9 0.19442 0.194 25.9 Abbreviations: SD,
standard deviation; VC, vehicle control (1% water/1% acetonitrile)
Note: For all calculations above, the resulting values are shown
with at least three significant figures for display purposes
only.
TABLE-US-00003 TABLE 3 Colchicine Effects on CYP2A6 Activity in
Pooled Human Liver Microsomes 7-Hydrxoycoumarin formation Specific
Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg protein)
Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean
.+-. SD of VC 0 0.47134 0.471 0.478 .+-. 0.00675 189 191 .+-. 2.70
100 (VC) 0.48746 0.487 195 0.47661 0.477 191 0.47708 0.477 191 0.2
0.44915 0.449 0.433 .+-. 0.0144 180 173 .+-. 5.75 90.5 0.42306
0.423 169 0.42562 0.426 170 2 0.48653 0.487 0.477 .+-. 0.00866 195
191 .+-. 3.47 99.7 0.47163 0.472 189 0.47142 0.471 189 10 0.44006
0.440 0.436 .+-. 0.00799 176 174 .+-. 3.20 91.2 0.44100 0.441 176
0.42671 0.427 171 20 0.44257 0.443 0.426 .+-. 0.0178 177 170 .+-.
7.12 89.1 0.42829 0.428 171 0.40719 0.407 163 50 0.43671 0.437
0.429 .+-. 0.00703 175 172 .+-. 2.81 89.8 0.42271 0.423 169 0.42865
0.429 171 Abbreviations: SD, standard deviation; VC, vehicle
control (1% water/1% acetonitrile) Note: For all calculations
above, the resulting values are shown with at least three
significant figures for display purposes only.
TABLE-US-00004 TABLE 4 Colchicine Effects on CYP2B6 Activity in
Pooled Human Liver Microsomes Nirvanol formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/mg protein) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC 0 0.19120 0.191 0.185 .+-. 0.00593 25.5 24.7 .+-. 0.791 100 (VC)
0.18768 0.188 25.0 0.18460 0.185 24.6 0.17726 0.177 23.6 0.2
0.14661 0.147 0.153 .+-. 0.00674 19.5 20.5 .+-. 0.899 82.8 0.16009
0.160 21.3 0.15348 0.153 20.5 2 0.19659 0.197 0.175 .+-. 0.0191
26.2 23.4 .+-. 2.54 94.7 0.15951 0.160 21.3 0.17025 0.170 22.7 10
0.16170 0.162 0.188 .+-. 0.0383 21.6 25.1 .+-. 5.10 102 0.23223
0.232 31.0 0.17127 0.171 22.8 20 0.16287 0.163 0.164 .+-. 0.00809
21.7 21.8 .+-. 1.08 88.4 0.17220 0.172 23.0 0.15609 0.156 20.8 50
0.17995 0.180 0.178 .+-. 0.00646 24.0 23.7 .+-. 0.861 96.0 0.18303
0.183 24.4 0.17063 0.171 22.8 Abbreviations: SD, standard
deviation; VC, vehicle control (1% water/1% acetonitrile) Note: For
all calculations above, the resulting values are shown with at
least three significant figures for display purposes only.
TABLE-US-00005 TABLE 5 Colchicine Effects on CYP2C8 Activity in
Pooled Human Liver Microsomes 6-Hydroxypaclitaxel formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg
protein) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC 0 0.13697 0.137 0.134 .+-. 0.00312 22.8 22.4
.+-. 0.520 100 (VC) 0.13730 0.137 22.9 0.13127 0.131 21.9 0.13229
0.132 22.0 0.2 0.11728 0.117 0.120 .+-. 0.00319 19.5 20.1 .+-.
0.532 89.5 0.12013 0.120 20.0 0.12365 0.124 20.6 2 0.12201 0.122
0.123 .+-. 0.00307 20.3 20.6 .+-. 0.512 91.8 0.12121 0.121 20.2
0.12689 0.127 21.1 10 0.12658 0.127 0.121 .+-. 0.00593 21.1 20.2
.+-. 0.989 90.3 0.11493 0.115 19.2 0.12270 0.123 20.5 20 0.12701
0.127 0.122 .+-. 0.00503 21.2 20.3 .+-. 0.838 90.8 0.12213 0.122
20.4 0.11695 0.117 19.5 50 0.11860 0.119 0.113 .+-. 0.00619 19.8
18.9 .+-. 1.03 84.3 0.10650 0.107 17.8 0.11484 0.115 19.1
Abbreviations: SD, standard deviation; VC, vehicle control (1%
water/1% acetonitrile) Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
TABLE-US-00006 TABLE 6 Colchicine Effects on CYP2C9 Activity in
Pooled Human Liver Microsomes 4'-Methylhydroxytolbutamide formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg
protein) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC 0 0.23379 0.234 0.165 .+-. 0.0543 62.3 44.1 .+-.
14.5 100 (VC) 0.12478 0.125 33.3 0.11865 0.119 31.6 0.18400 0.184
49.1 0.2 0.13035 0.130 0.116 .+-. 0.0181 34.8 30.8 .+-. 4.82 69.9
0.12104 0.121 32.3 0.09543 0.0954 25.4 2 0.27491 0.275 0.254 .+-.
0.0185 73.3 67.8 .+-. 4.94 154 0.24949 0.249 66.5 0.23889 0.239
63.7 10 0.15320 0.153 0.133 .+-. 0.0205 40.9 35.5 .+-. 5.46 80.6
0.11226 0.112 29.9 0.13406 0.134 35.7 20 0.17773 0.178 0.158 .+-.
0.0216 47.4 42.2 .+-. 5.75 95.8 0.16209 0.162 43.2 0.13510 0.135
36.0 50 0.16757 0.168 0.147 .+-. 0.0181 44.7 39.3 .+-. 4.82 89.2
0.14235 0.142 38.0 0.13254 0.133 35.3 Abbreviations: SD, standard
deviation; VC, vehicle control (1% water/1% acetonitrile) Note: For
all calculations above, the resulting values are shown with at
least three significant figures for display purposes only.
TABLE-US-00007 TABLE 7 Colchicine Effects on CYP2C19 Activity in
Pooled Human Liver Microsomes 4'-Hydroxymephenytoin formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg
protein) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC 0 0.09628 0.0963 0.0920 .+-. 0.00522 12.8 12.3
.+-. 0.696 100 (VC) 0.09414 0.0941 12.6 0.09304 0.0930 12.4 0.08440
0.0844 11.3 0.2 0.08140 0.0814 0.0840 .+-. 0.00266 10.9 11.2 .+-.
0.355 91.3 0.08384 0.0838 11.2 0.08672 0.0867 11.6 2 0.09459 0.0946
0.0986 .+-. 0.00359 12.6 13.1 .+-. 0.479 107 0.10150 0.102 13.5
0.09975 0.0998 13.3 10 0.08531 0.0853 0.0856 .+-. 0.00226 11.4 11.4
.+-. 0.301 93.1 0.08351 0.0835 11.1 0.08800 0.0880 11.7 20 0.09475
0.0948 0.0953 .+-. 0.000979 12.6 12.7 .+-. 0.131 104 0.09641 0.0964
12.9 0.09468 0.0947 12.6 50 0.09257 0.0926 0.0927 .+-. 0.00115 12.3
12.4 .+-. 0.153 101 0.09156 0.0916 12.2 0.09385 0.0939 12.5
Abbreviations: SD, standard deviation; VC, vehicle control (1%
water/1% acetonitrile) Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
TABLE-US-00008 TABLE 8 Colchicine Effects on CYP2D6 Activity in
Pooled Human Liver Microsomes Dextrorphan formation Specific
Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg protein)
Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean
.+-. SD of VC 0 0.06696 0.0670 0.0616 .+-. 0.00836 17.9 16.4 .+-.
2.23 100 (VC) 0.06211 0.0621 16.6 0.06770 0.0677 18.1 0.04962
0.0496 13.2 0.2 0.06445 0.0645 0.0664 .+-. 0.00171 17.2 17.7 .+-.
0.457 108 0.06687 0.0669 17.8 0.06776 0.0678 18.1 2 0.07072 0.0707
0.0691 .+-. 0.00296 18.9 18.4 .+-. 0.790 112 0.07097 0.0710 18.9
0.06572 0.0657 17.5 10 0.06348 0.0635 0.0647 .+-. 0.00189 16.9 17.3
.+-. 0.503 105 0.06383 0.0638 17.0 0.06691 0.0669 17.8 20 0.07091
0.0709 0.0733 .+-. 0.00230 18.9 19.5 .+-. 0.614 119 0.07350 0.0735
19.6 0.07550 0.0755 20.1 50 0.06545 0.0655 0.0664 .+-. 0.00166 17.5
17.7 .+-. 0.442 108 0.06535 0.0654 17.4 0.06827 0.0683 18.2
Abbreviations: SD, standard deviation; VC, vehicle control (1%
water/1% acetonitrile) Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
TABLE-US-00009 TABLE 9 Colchicine Effects on CYP2E1 Activity in
Pooled Human Liver Microsomes 6-Hydroxychlorzoxazone formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg
protein) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC 0 0.81146 0.811 0.787 .+-. 0.0207 216 210 .+-.
5.53 100 (VC) 0.76298 0.763 203 0.77971 0.780 208 0.79492 0.795 212
0.2 0.76699 0.767 0.749 .+-. 0.0310 205 200 .+-. 8.27 95.2 0.76713
0.767 205 0.71336 0.713 190 2 0.81289 0.813 0.780 .+-. 0.0287 217
208 .+-. 7.65 99.1 0.76807 0.768 205 0.75942 0.759 203 10 0.72994
0.730 0.739 .+-. 0.00901 195 197 .+-. 2.40 93.9 0.74788 0.748 199
0.74045 0.740 197 20 0.79882 0.799 0.798 .+-. 0.0122 213 213 .+-.
3.26 101 0.81021 0.810 216 0.78576 0.786 210 50 0.78319 0.783 0.756
.+-. 0.0259 209 202 .+-. 6.91 96.0 0.73161 0.732 195 0.75330 0.753
201 Abbreviations: SD, standard deviation; VC, vehicle control (1%
water/1% acetonitrile) Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
TABLE-US-00010 TABLE 10 Colchicine Effects on CYP3A4 Activity in
Pooled Human Liver Microsomes 6.beta.-Hydroxytestosterone formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/mg
protein) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC 0 0.43411 0.434 0.452 .+-. 0.0126 347 362 .+-.
10.1 100 (VC) 0.46351 0.464 371 0.45573 0.456 365 0.45533 0.455 364
0.2 0.52049 0.520 0.539 .+-. 0.0162 416 431 .+-. 13.0 119 0.55077
0.551 441 0.54570 0.546 437 2 0.56131 0.561 0.559 .+-. 0.0147 449
447 .+-. 11.8 124 0.54295 0.543 434 0.57211 0.572 458 10 0.57041
0.570 0.543 .+-. 0.0262 456 435 .+-. 20.9 120 0.51812 0.518 414
0.54193 0.542 434 20 0.56052 0.561 0.786 .+-. 0.271 448 629 .+-.
217 174 1.08621 1.09 869 0.71114 0.711 569 50 0.74399 0.744 0.685
.+-. 0.0511 595 548 .+-. 40.9 151 0.65574 0.656 525 0.65520 0.655
524 Abbreviations: SD, standard deviation; VC, vehicle control (1%
water/1% acetonitrile) Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
[0181] Under these experimental conditions, no tested concentration
of colchicine inhibited activity of CYP1A2 (Table 2), CYP2B6 (Table
4), CYP2C9 (Table 6), CYP2C19 (Table 7), CYP2D6 (Table 8), CYP2E1
(Table 9), or CYP3A4 (Table 10) in human liver microsomes at a
statistically significant level (p>0.05 using an unpaired
two-tailed t-test).
[0182] However, under these experimental conditions, colchicine did
inhibit activities of CYP2A6 (Table 3) and CYP2C8 (Table 5) in
human liver microsomes at one or more of the tested colchicine
concentrations at a statistically significant level (p.ltoreq.0.05
using an unpaired two-tailed t-test). IC.sub.50 values were greater
than 50 .mu.M.
[0183] Additionally, under these experimental conditions,
colchicine activated activity of CYP3A4 (Table 10) in human liver
microsomes at one or more of the tested colchicine concentrations
at a statistically significant level (p.ltoreq.0.05 using an
unpaired two-tailed t-test). The maximum activity observed was 174%
of the control activity.
Example 2
Colchicine Induction of Cytochrome P450 Isozymes
[0184] The study of this example was performed to determine if
there is induction or suppression of cytochrome P450 isozymes
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1,
and CYP3A4 in human hepatocytes following in vitro exposure to
colchicine. These induction/inhibition studies used cryopreserved
human hepatocytes and compared enzymatic activity levels for each
of these cytochrome P450 isozymes, using an appropriate enzyme
substrate, in the human hepatocytes following in vitro exposure for
48.+-.3 hrs to the presence or absence of colchicine.
[0185] Hepatocytes from three human donors were obtained from a
cryopreserved hepatocyte bank (In Vitro Technologies, Inc.,
USA).
[0186] Donor 1 was reported to be a 40-year old Caucasian female
who died of an accidental drug overdose, with a medical history
including hypertension. Serology testing was negative except for
cytomegalovirus. Donor 1 had a history of tobacco use ("half-pack
per day for 20 years") and drug abuse (cocaine, crack, crank,
prescription drugs and marijuana). Recreational medications listed
were LIBRIUM, LORTAB, and ATIVAN.
[0187] Donor 2 was reported to be a 51-year old Caucasian male who
died of ischemic stroke, with a medical history including diabetes,
hypertension, kidney stone removal, sleep apnea, depression/anxiety
and colitis. No chronic medications were listed. Serology testing
was negative except for cytomegalovirus. Donor 2 was known to smoke
tobacco ("half-pack per day for 20 years"); alcohol and narcotic
and cannabinoid use by Donor 2 reportedly ceased 15 years prior to
donation.
[0188] Donor 3 was reported to be a 54-year old Caucasian female
who died of cardiac arrest, with a medical history including high
cholesterol. No chronic medications were reported Serology testing
was negative, including cytomegalovirus. Donor 3 was known to smoke
tobacco ("one-pack per day for 35 years"). No history of alcohol or
other drug use.
[0189] After thawing, viable hepatocytes from each donor were
transferred to collagen-coated 48-well plates for attachment in
plating medium (DMEM stock (Dulbecco's modified Eagle's medium,
supplemented with bovine serum albumin, fructose,
N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonate) (HEPES), and
sodium bicarbonate), supplemented with antibiotics, bovine serum,
hydrocortisone, insulin and minimum essential medium (MEM)
nonessential amino acids). After attachment to the collagen matrix,
plating medium was replaced with sandwich medium (incubation medium
supplemented with VITROGEN and incubated until use. All incubations
were conducted at 37.+-.1.degree. C., 95% air/5% CO2 and saturating
humidity.
[0190] After establishment of the hepatocyte cultures, sandwich
medium was removed and the hepatocytes were incubated with
incubation solution (DMEM stock supplemented with antibiotics,
hydrocortisone, insulin, and MEM non-essential amino acids)
containing 0.25, 2.5, or 25 .mu.M colchicine for 24.+-.1.5 hrs.
Incubation solution was aspirated and replaced with incubation
solution containing the same concentration of colchicine and
incubated for an additional 24.+-.1.5 hrs. After the colchicine
treatment period, the incubation solution was replaced with 150
.mu.L Krebs-Henseleit (KHB) buffer supplemented with antibiotics,
calcium chloride, heptanoic acid, HEPES, and sodium bicarbonate
(supplemented KHB) and incubated for 10 minutes. The supplemented
KHB was replaced with 150 .mu.L supplemented KHB containing the
appropriate isoform-selective substrate and incubated for 4 hrs
prior to termination by adding 150 .mu.L ice-cold methanol, except
for the CYP2C8 incubations which were terminated by adding 150
.mu.L acetonitrile. Samples were transferred to cryovials and
analyzed after storage at -70.degree. C. Three replicates were
performed at each colchicine concentration for each cytochrome P450
isozyme.
[0191] Analogous vehicle control experiments were also performed to
establish a baseline value for enzyme activity in the absence of
colchicine. Vehicle control experiments were performed as described
above for the test incubations, except that the incubation medium
included no colchicine. Four replicates were performed of the
vehicle control for each donor.
[0192] A table of the substrate, substrate concentration,
metabolite formed, and metabolite assay method for each CYP isozyme
studied is provided below. All substrates were dissolved in
acetonitrile as 100.times. solutions. All 100.times. substrate
solutions were diluted with supplemented KHB to the final
concentrations listed below, except for paclitaxel, which was
diluted with incubation medium.
TABLE-US-00011 TABLE 11 Isoform-selective substrates for cytochrome
P450 isozymes in the colchicine induction study. Isoform-selective
Substrate Metabolite CYP isoform substrate concentration Metabolite
formed Assay CYP1A2 Phenacetin 100 .mu.M acetaminophen LC/MS CYP2A6
Coumarin 100 .mu.M 7-hydroxycoumarin, HPLC-UV 7-hydroxy coumarin
glucuronide, 7-hydroxycoumarin sulfate CYP2B6 S-Mephenytoin 1 mM
nirvanol LC/MS CYP2C8 Paclitaxel 50 .mu.M 6-hydroxy paclitaxel
HPLC-UV CYP2C9 Tolbutamide 50 .mu.M 4'-methylhydroxytolbutamide
LC/MS CYP2C19 S-Mephenytoin 100 .mu.M 4'-hydroxy mephenytoin LC/MS
CYP2D6 Dextromethorphan 16 .mu.M dextrorphan LC/MS CYP2E1
Chlorzoxazone 300 .mu.M 6-hydroxychlorzoxazone LC/MS CYP3A4
Testosterone 125 .mu.M 6.beta.-hydroxy testosterone HPLC-UV
[0193] Colchicine 100.times. stock solutions were prepared in water
as described above in Example 1 and diluted with incubation medium
and acetonitrile to produce incubation solutions with final
concentrations of 0.25, 2.5, and 25 .mu.M colchicine, each
containing 1% water and 1% acetonitrile.
[0194] Positive controls (n=4) were performed to verify that the
test system was sensitive to known inducers by testing induction of
CYP1A2 and CYP3A4 by 50CM omeprazole and 25 M rifampicin,
respectively, using the appropriate isoform-selective enzyme
substrate. Following treatment with 50 .mu.M omeprazole, CYP1A2
activity was 653%, 765%, and 596% of the vehicle control in human
hepatocytes prepared from Donors 1, 2, and 3, respectively.
Following treatment with 25 .mu.M rifampin, CYP3A4 activity was
2,796%, >2,092%, and 2,633% of the VC in human hepatocytes
prepared from Donors 1, 2, and 3, respectively. Based on these
increases in activities of CYP1A2 and CYP3A4 following treatment
with the known inducers; the hepatocytes from the three donors were
considered inducible.
[0195] Additionally, reference control samples were included to
evaluate inducibility of CYP2B6, CYP2C8, CYP2C9, and CYP2C19 in the
test system. The reference controls included 1 mM phenobarbital
(for CYP2B6) or 25 .mu.M rifampicin as the reference inducer. The
reference controls showed a statistically significant amount of
induction for each hepatocyte donor for CYP2C9, although the amount
of induction varied between the three hepatocyte donors (299%,
306%, and 279% for donors 1, 2 and 3, respectively). For CYP2B6,
phenobarbital-induced activity in donors 1, 2 and, 3 were 757%,
639%, and 419%, respectively. The induction for Donor 3 was
calculated with the measured amounts of nirvanol formed, even
thought the amount was less than the lower limit of quantitation
(LLOQ) for the compound in each replicate of the vehicle control
and in two of the four replicates of the rifampicin reference
control. The reference controls for CYP2C8 showed a statistically
significant amount of induction for each hepatocyte donor, although
the amount of induction varied between the three hepatocyte donors
and the measured amounts of 6-hydroxypaclitaxel formed were
generally less than the lower limit of quantitation (LLOQ) for
6-hydroxypaclitaxel. For CYP2C19, rifampin induced activity in
donors 1 (317%), 2 (247%) and 3 (277%). The induction of CYP2C19 by
rifampicin was calculated with the measured amounts of
4'-hydroxymephenyloin formed, even though the amount was less than
the lower limit of quantitation (LLOQ) for the compound in each
replicate of the vehicle controls and the rifampicin reference
controls. Therefore, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 in the
hepatocytes from these donors were considered induced by rifampin
and phenobarbital.
[0196] Furthermore, interference controls were performed for each
CYP isozyme to determine whether or not colchicine or its
metabolites interfered with detection of the isoform-specific
metabolites. In these controls, performed in duplicate, the
hepatocytes were incubated with colchicine as for the test samples,
and then incubated with the buffer of the isoform-specific
substrate (without substrate) as for the test samples. No
interference of colchicine or its metabolite was observed in any of
the assays for detection of the isoform-specific metabolites formed
in the test systems.
[0197] Results for each cytochrome P450 isozyme are shown in Tables
12-20. Induction was not observed at these experimental conditions
for any of the tested isozymes: CYP1A2, CYP2A6, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. However, statistically
significant inhibition in enzyme activity was observed for each of
the nine CYPS studied. Statistical significance of a change in
specific activity from that measured for the vehicle control (0
.mu.M colchicine) was determined using a two-tailed t-test. Mean
specific activity values with associated p-values.ltoreq.0.05 were
deemed to be statistically significant.
TABLE-US-00012 TABLE 12 CYP1A2 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Acetaminophen formation Specific
Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells)
Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean
.+-. SD of VC Human Donor 1 0 0.18064 0.181 0.177 .+-. 0.00766 1.61
1.58 .+-. 0.0684 100 (VC) 0.16701 0.167 1.49 0.17518 0.175 1.56
0.18473 0.185 1.65 0.25 0.03089 0.0309 0.0406 .+-. 0.0121 0.276
0.362 .+-. 0.108 22.9 0.03671 0.0367 0.328 0.05413 0.0541 0.483 2.5
0.04389 0.0439 0.0459 .+-. 0.00183 0.392 0.410 .+-. 0.0163 26.0
0.04744 0.0474 0.424 0.04640 0.0464 0.414 25 0.04071 0.0407 0.0415
.+-. 0.00248 0.363 0.371 .+-. 0.0222 23.5 0.04433 0.0443 0.396
0.03958 0.0396 0.353 Human Donor 2 0 0.22463 0.225 0.236 .+-.
0.01034 2.01 2.11 .+-. 0.0923 100 (VC) 0.07823* N/A N/A 0.23985
0.240 2.14 0.24437 0.244 2.18 0.25 0.04672 0.0467 0.0462 .+-.
0.00145 0.417 0.413 .+-. 0.0130 19.6 0.04732 0.0473 0.423 0.04456
0.0446 0.398 2.5 0.04696 0.0470 0.0488 .+-. 0.00234 0.419 0.436
.+-. 0.0209 20.7 0.04812 0.0481 0.430 0.05146 0.0515 0.459 25
0.03998 0.0400 0.0423 .+-. 0.00207 0.357 0.378 .+-. 0.0185 17.9
0.04382 0.0438 0.391 0.04323 0.0432 0.386 Human Donor 3 0 0.72070
0.721 0.757 .+-. 0.0439 6.43 6.76 .+-. 0.392 100 (VC) 0.71756 0.718
6.41 0.79898 0.799 7.13 0.79073 0.791 7.06 0.25 0.15270 0.153 0.180
.+-. 0.0236 1.36 1.61 .+-. 0.211 23.8 0.19188 0.192 1.71 0.19501
0.195 1.74 2.5 0.16389 0.164 0.180 .+-. 0.0173 1.46 1.61 .+-. 0.155
23.8 0.17886 0.179 1.60 0.19841 0.198 1.77 25 0.15280 0.153 0.157
.+-. 0.00699 1.36 1.40 .+-. 0.0625 20.7 0.16497 0.165 1.47 0.15291
0.153 1.37 Abbreviations: SD, standard deviation; VC, vehicle
control (1% water/1% acetonitrile); N/A, not applicable *Sample raw
data value will be excluded from all calculations due to low
cellular confluence observed during incubation. Note: For all
calculations above, the resulting values are shown with at least
three significant figures for display purposes only.
TABLE-US-00013 TABLE 13a CYP2A6 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Metabolite formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC 7-Hydroxycoumarin (7-HC) Formation: Human Donor 1 0
0.05799.sup.a <0.100 <0.100 .+-. 0.000 <0.893 <0.893
.+-. 0.000 100 (VC) 0.05039.sup.a <0.100 <0.893 0.06564.sup.a
<0.100 <0.893 0.03394.sup.a <0.100 <0.893 0.25
0.00000.sup.a <0.100 <0.100 .+-. 0.000 <0.893 <0.893
.+-. 0.000 100 0.00000.sup.a <0.100 <0.893 0.00000.sup.a
<0.100 <0.893 2.5 0.00000.sup.a <0.100 <0.100 .+-.
0.000 <0.893 <0.893 .+-. 0.000 100 0.00000.sup.a <0.100
<0.893 0.00000.sup.a <0.100 <0.893 25 0.00000.sup.a
<0.100 <0.100 .+-. 0.000 <0.893 <0.893 .+-. 0.000 100
0.00000.sup.a <0.100 <0.893 0.00000.sup.a <0.100 <0.893
7-Hydroxycoumarin (7-HC) Formation: Human Donor 2 0 0.00000.sup.a
<0.100 <0.100 .+-. 0.000 <0.893 <0.893 .+-. 0.000 100
(VC) 0.07417.sup.a <0.100 <0.893 0.00000.sup.a <0.100
<0.893 0.06269.sup.a <0.100 <0.893 0.25 0.06360.sup.a
<0.100 <0.100 .+-. 0.000 <0.893 <0.893 .+-. 0.000 100
0.05338.sup.a <0.100 <0.893 0.00000.sup.a <0.100 <0.893
2.5 0.00000.sup.a <0.100 <0.100 .+-. 0.000 <0.893
<0.893 .+-. 0.000 100 0.00000.sup.a <0.100 <0.893
0.04979.sup.a <0.100 <0.893 25 0.00000.sup.a <0.100
<0.100 .+-. 0.000 <0.893 <0.893 .+-. 0.000 100
0.00000.sup.a <0.100 <0.893 0.00000.sup.a <0.100 <0.893
7-Hydroxycoumarin (7-HC) Formation: Human Donor 3 0 0.11042 0.110
<0.104 .+-. 0.00501 0.986 <0.928 .+-. 0.0447 100 (VC)
0.00000.sup.a <0.100 <0.893 0.05331.sup.a <0.100 <0.893
0.10546 0.105 0.942 0.25 0.10795 0.108 <0.103 .+-. 0.00459 0.964
<0.917 .+-. 0.0410 98.7 0.09518.sup.a <0.100 <0.893
0.08086.sup.a <0.100 <0.893 2.5 0.08970.sup.a <0.100
<0.100 .+-. 0.0000981 <0.893 <0.893 .+-. 0.000876 96.2
0.10017 0.100 0.894 0.08526.sup.a <0.100 <0.893 25 0.19516
0.195 <0.133 .+-. 0.0541 1.74 <1.18 .+-. 0.483 128
0.08873.sup.a <0.100 <0.893 0.10294 0.103 0.919
Abbreviations: SD, standard deviation; VC, vehicle control (1%
water/1% acetonitrile) .sup.aThe observed analyzed value (.mu.M)
was below the lowest concentration on the standard curve (0.1
.mu.M). Note: For all calculations above, the resulting values are
shown with at least three significant figures for display purposes
only.
TABLE-US-00014 TABLE 13b CYP2A6 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Metabolite formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC 7-Hydroxycoumarin Glucuronide (7-HCG) Formation: Human Donor 1 0
1.32662 1.33 1.16 .+-. 0.196 11.8 10.4 .+-. 1.75 100 (VC) 1.26876
1.27 11.3 1.17175 1.17 10.5 0.88485 0.885 7.90 0.25 0.21711 0.217
0.220 .+-. 0.00800 1.94 1.96 .+-. 0.0715 18.9 0.21379 0.214 1.91
0.22901 0.229 2.04 2.5 0.23406 0.234 0.240 .+-. 0.0125 2.09 2.14
.+-. 0.112 20.6 0.23129 0.231 2.07 0.25418 0.254 2.27 25 0.18343
0.183 0.148 .+-. 0.0322 1.64 1.32 .+-. 0.287 12.7 0.13977 0.140
1.25 0.12069 0.121 1.08 7-Hydroxycoumarin Glucuronide (7-HCG)
Formation: Human Donor 2 0 0.11407 0.114 0.102 .+-. 0.0121 1.02
0.908 .+-. 0.108 100 (VC) 0.10862 0.109 0.970 0.09688 0.0969 0.865
0.08699 0.0870 0.777 0.25 0.00000.sup.a <0.0500 <0.0500 .+-.
0.000 <0.446 <0.446 .+-. 0.000 <49.2 0.00000.sup.a
<0.0500 <0.446 0.00000.sup.a <0.0500 <0.446 2.5
0.01661.sup.a <0.0500 <0.0500 .+-. 0.000 <0.446 <0.446
.+-. 0.000 <49.2 0.00000.sup.a <0.0500 <0.446
0.00000.sup.a <0.0500 <0.446 25 0.00000.sup.a <0.0500
<0.0500 .+-. 0.000 <0.446 <0.446 .+-. 0.000 <49.2
0.00000.sup.a <0.0500 <0.446 0.00000.sup.a <0.0500
<0.446 7-Hydroxycoumarin Glucuronide (7-HCG) Formation: Human
Donor 3 0 1.47077 1.47 1.49 .+-. 0.0564 13.1 13.3 .+-. 0.504 100
(VC) 1.48203 1.48 13.2 1.42782 1.43 12.7 1.56287 1.56 14.0 0.25
0.92785 0.928 0.722 .+-. 0.195 8.28 6.44 .+-. 1.74 48.6 0.69597
0.696 6.21 0.54111 0.541 4.83 2.5 0.67426 0.674 0.780 .+-. 0.101
6.02 6.97 .+-. 0.906 52.5 0.79062 0.791 7.06 0.87632 0.876 7.82 25
0.92590 0.926 0.772 .+-. 0.135 8.27 6.89 .+-. 1.20 52.0 0.71307
0.713 6.37 0.67693 0.677 6.04 Abbreviations: SD, standard
deviation; VC, vehicle control (1% water/1% acetonitrile) .sup.aThe
observed analyzed value (.mu.M) was below the lowest concentration
on the standard curve (0.05 .mu.M). Note: For all calculations
above, the resulting values are shown with at least three
significant figures for display purposes only.
TABLE-US-00015 TABLE 13c CYP2A6 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Metabolite formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC 7-Hydroxycoumarin Sulfate (7-HCS) Formation: Human Donor 1 0
0.13390.sup.a <0.150 <0.150 .+-. 0.000 <1.34 <1.34 .+-.
0.000 100 (VC) 0.13422.sup.a <0.150 <1.34 0.12167.sup.a
<0.150 <1.34 0.10325.sup.a <0.150 <1.34 0.25
0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34 <1.34 .+-.
0.000 100 0.00000.sup.a <0.150 <1.34 0.00000.sup.a <0.150
<1.34 2.5 0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34
<1.34 .+-. 0.000 100 0.00000.sup.a <0.150 <1.34
0.00000.sup.a <0.150 <1.34 25 0.00000.sup.a <0.150
<0.150 .+-. 0.000 <1.34 <1.34 .+-. 0.000 100 0.00000.sup.a
<0.150 <1.34 0.00000.sup.a <0.150 <1.34
7-Hydroxycoumarin Sulfate (7-HCS) Formation: Human Donor 2 0
0.05679.sup.a <0.150 <0.150 .+-. 0.000 <1.34 <1.34 .+-.
0.000 100 (VC) 0.05088.sup.a <0.150 <1.34 0.00000.sup.a
<0.150 <1.34 0.00000.sup.a <0.150 <1.34 0.25
0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34 <1.34 .+-.
0.000 100 0.00000.sup.a <0.150 <1.34 0.00000.sup.a <0.150
<1.34 2.5 0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34
<1.34 .+-. 0.000 100 0.00000.sup.a <0.150 <1.34
0.00000.sup.a <0.150 <1.34 25 0.00000.sup.a <0.150
<0.150 .+-. 0.000 <1.34 <1.34 .+-. 0.000 100 0.00000.sup.a
<0.150 <1.34 0.00000.sup.a <0.150 <1.34
7-Hydroxycoumarin Sulfate (7-HCS) Formation: Human Donor 3 0
0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34 <1.34 .+-.
0.000 100 (VC) 0.00000.sup.a <0.150 <1.34 0.00000.sup.a
<0.150 <1.34 0.00000.sup.a <0.150 <1.34 0.25
0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34 <1.34 .+-.
0.000 100 0.00000.sup.a <0.150 <1.34 0.00000.sup.a <0.150
<1.34 2.5 0.00000.sup.a <0.150 <0.150 .+-. 0.000 <1.34
<1.34 .+-. 0.000 100 0.00000.sup.a <0.150 <1.34
0.00000.sup.a <0.150 <1.34 25 0.00000.sup.a <0.150
<0.150 .+-. 0.000 <1.34 <1.34 .+-. 0.000 100 0.00000.sup.a
<0.150 <1.34 0.00000.sup.a <0.150 <1.34 Abbreviations:
SD, standard deviation; VC, vehicle control (1% water/1%
acetonitrile) .sup.aThe observed analyzed value (.mu.M) was below
the lowest concentration on the standard curve (0.15 .mu.M). Note:
For all calculations above, the resulting values are shown with at
least three significant figures for display purposes only.
TABLE-US-00016 TABLE 13D CYP2A6 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Metabolite formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC Total Metabolite Formation: Human Donor 1 0 1.52.sup.a <1.58
<1.41 .+-. 0.196 <14.1 <12.6 .+-. 1.75 100 (VC) 1.45.sup.a
<1.52 <13.6 1.36.sup.a <1.42 <12.7 1.02.sup.a <1.13
<10.1 0.25 0.217.sup.a <0.467 <0.470 .+-. 0.00800 <4.17
<4.20 .+-. 0.0715 33.3 0.214.sup.a <0.464 <4.14
0.229.sup.a <0.479 <4.28 2.5 0.234.sup.a <0.484 <0.490
.+-. 0.0125 <4.32 <4.37 .+-. 0.112 34.7 0.231.sup.a <0.481
<4.30 0.254.sup.a <0.504 <4.50 25 0.183.sup.a <0.433
<0.398 .+-. 0.0322 <3.87 <3.55 .+-. 0.287 28.2 0.140.sup.a
<0.390 <3.48 0.121.sup.a <0.371 <3.31 Total Metabolite
Formation: Human Donor 2 0 0.171.sup.a <0.364 <0.352 .+-.
0.0121 <3.25 <3.14 .+-. 0.108 100 (VC) 0.234.sup.a <0.359
<3.20 0.0969.sup.a <0.347 <3.10 0.150.sup.a <0.337
<3.01 0.25 0.0636.sup.b <0.300 <0.300 .+-. 0.000 <2.68
<2.68 .+-. 0.000 85.3 0.0534.sup.b <0.300 <2.68
0.000.sup.b <0.300 <2.68 2.5 0.0166.sup.b <0.300 <0.300
.+-. 0.000 <2.68 <2.68 .+-. 0.000 85.3 0.000.sup.b <0.300
<2.68 0.0498.sup.b <0.300 <2.68 25 0.000.sup.b <0.300
<0.300 .+-. 0.000 <2.68 <2.68 .+-. 0.000 85.3 0.000.sup.b
<0.300 <2.68 0.000.sup.b <0.300 <2.68 Total Metabolite
Formation: Human Donor 3 0 1.58.sup.c <1.73 <1.74 .+-. 0.0581
<15.5 <15.5 .+-. 0.519 100 (VC) 1.48.sup.a <1.73 <15.5
1.48.sup.a <1.68 <15.0 1.67.sup.c <1.82 <16.2 0.25
1.04.sup.c <1.19 <0.974 .+-. 0.199 <10.6 <8.70 .+-.
1.78 56.0 0.791.sup.a <0.946 <8.45 0.622.sup.a <0.791
<7.06 2.5 0.764.sup.a <0.924 <1.03 .+-. 0.101 <8.25
<9.20 .+-. 0.906 59.2 0.891.sup.c <1.04 <9.29 0.962.sup.a
<1.13 <10.1 25 1.12.sup.c <1.27 <1.05 .+-. 0.188
<11.3 <9.42 .+-. 1.68 60.6 0.802.sup.a <0.963 <8.60
0.780.sup.c <0.930 <8.30 Abbreviations: SD, standard
deviation; VC, vehicle control (1% water/1% acetonitrile) .sup.aThe
observed analyzed value (.mu.M) for the 7-HC & 7-HCS
metabolites were below the lowest concentration on the
corresponding standard curve. .sup.bThe observed analyzed value
(.mu.M) for all metabolites were below the lowest concentration on
the corresponding standard curve. .sup.cThe observed analyzed value
(.mu.M) for the 7-HCS metabolite was below the lowest concentration
on the standard curve. Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
TABLE-US-00017 TABLE 14 CYP2B6 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Nirvanol formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC Human Donor 1 0 0.07114 0.0711 0.0737 .+-. 0.00204 0.635 0.658
.+-. 0.0183 100 (VC) 0.07302 0.0730 0.652 0.07575 0.0758 0.676
0.07485 0.0749 0.668 0.25 0.03628 0.0363 0.0362 .+-. 0.0000781
0.324 0.323 .+-. 0.000697 49.2 0.03627 0.0363 0.324 0.03614 0.0361
0.323 2.5 0.03089 0.0309 0.0357 .+-. 0.00414 0.276 0.318 .+-.
0.0370 48.4 0.03834 0.0383 0.342 0.03776 0.0378 0.337 25 0.03477
0.0348 0.0356 .+-. 0.00260 0.310 0.318 .+-. 0.0232 48.3 0.03347
0.0335 0.299 0.03848 0.0385 0.344 Human Donor 2 0 0.06940 0.0694
0.0850 .+-. 0.0105 0.620 0.759 .+-. 0.0941 100 (VC) 0.09237 0.0924
0.825 0.08983 0.0898 0.802 0.08852 0.0885 0.790 0.25 0.04447 0.0445
0.0420 .+-. 0.00424 0.397 0.375 .+-. 0.0378 49.3 0.03706 0.0371
0.331 0.04432 0.0443 0.396 2.5 0.04629 0.0463 0.0426 .+-. 0.00385
0.413 0.380 .+-. 0.0343 50.1 0.03861 0.0386 0.345 0.04283 0.0428
0.382 25 0.05201 0.0520 0.0510 .+-. 0.00314 0.464 0.455 .+-. 0.0281
60.0 0.04747 0.0475 0.424 0.05351 0.0535 0.478 Human Donor 3 0
0.00601.sup.a <0.0250 <0.0250 .+-. 0.000 <0.223 <0.223
.+-. 0.000 100 (VC) 0.00609.sup.a <0.0250 <0.223
0.00681.sup.a <0.0250 <0.223 0.00666.sup.a <0.0250
<0.223 0.25 0.00511.sup.a <0.0250 <0.0250 .+-. 0.000
<0.223 <0.223 .+-. 0.000 100 0.00502.sup.a <0.0250
<0.223 0.00508.sup.a <0.0250 <0.223 2.5 0.00543.sup.a
<0.0250 <0.0250 .+-. 0.000 <0.223 <0.223 .+-. 0.000 100
0.00527.sup.a <0.0250 <0.223 0.00579.sup.a <0.0250
<0.223 25 0.00569.sup.a <0.0250 <0.0250 .+-. 0.000
<0.223 <0.223 .+-. 0.000 100 0.00600.sup.a <0.0250
<0.223 0.00535.sup.a <0.0250 <0.223 Abbreviations: SD,
standard deviation; VC, vehicle control (1% water/1% acetonitrile)
.sup.aThe observed analyzed value (.mu.M) was below the lowest
concentration on the standard curve (0.025 .mu.M). Note: For all
calculations above, the resulting values are shown with at least
three significant figures for display purposes only.
TABLE-US-00018 TABLE 15 CYP2C8 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition 6-Hydroxypaclitaxel formation Specific
Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells)
Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean
.+-. SD of VC Human Donor 1 0 0.02374.sup.a <0.0500 <0.0500
.+-. 0.0000 <0.446 <0.446 .+-. 0.000 100 (VC) 0.02292.sup.a
<0.0500 <0.446 0.01719.sup.a <0.0500 <0.446
0.01654.sup.a <0.0500 <0.446 0.25 0.00815.sup.a <0.0500
<0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000 100
0.00808.sup.a <0.0500 <0.446 (39.7) 0.00769.sup.a <0.0500
<0.446 2.5 0.00823.sup.a <0.0500 <0.0500 .+-. 0.0000
<0.446 <0.446 .+-. 0.000 100 0.00802.sup.a <0.0500
<0.446 (40.5) 0.00815.sup.a <0.0500 <0.446 25
0.00781.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446
.+-. 0.000 100 0.00748.sup.a <0.0500 <0.446 (38.8)
0.00810.sup.a <0.0500 <0.446 Human Donor 2 0 0.01378.sup.a
<0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000
100 (VC) 0.01243.sup.a <0.0500 <0.446 0.01247.sup.a
<0.0500 <0.446 0.01208.sup.a <0.0500 <0.446 0.25
0.00000.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446
.+-. 0.000 100 0.00000.sup.a <0.0500 <0.446 0.00000.sup.a
<0.0500 <0.446 2.5 0.00000.sup.a <0.0500 <0.0500 .+-.
0.0000 <0.446 <0.446 .+-. 0.000 100 0.00000.sup.a <0.0500
<0.446 0.00000.sup.a <0.0500 <0.446 25 0.00000.sup.a
<0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000
100 0.00000.sup.a <0.0500 <0.446 0.00000.sup.a <0.0500
<0.446 Human Donor 3 0 0.02195.sup.a <0.0500 <0.0500 .+-.
0.0000 <0.446 <0.446 .+-. 0.000 100 (VC) 0.02203.sup.a
<0.0500 <0.446 0.02217.sup.a <0.0500 <0.446
0.02098.sup.a <0.0500 <0.446 0.25 0.00899.sup.a <0.0500
<0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000 100
0.00910.sup.a <0.0500 <0.446 (41.3) 0.00887.sup.a <0.0500
<0.446 2.5 0.00913.sup.a <0.0500 <0.0500 .+-. 0.0000
<0.446 <0.446 .+-. 0.000 100 0.00924.sup.a <0.0500
<0.446 (41.9) 0.00901.sup.a <0.0500 <0.446 25
0.00869.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446
.+-. 0.000 100 0.00864.sup.a <0.0500 <0.446 (39.7)
0.00863.sup.a <0.0500 <0.446 Abbreviations: SD, standard
deviation; VC, vehicle control (1% water/1% acetonitrile) .sup.aThe
observed analyzed value (.mu.M) was below the lowest concentration
on the standard curve (0.05 .mu.M). Note: For all calculations
above, the resulting values are shown with at least three
significant figures for display purposes only.
TABLE-US-00019 TABLE 16 CYP2C9 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition 4'-Methylhydroxytolbutamide formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/million
cells) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC Human Donor 1 0 0.13088 0.131 0.157 .+-. 0.0176
1.17 1.40 .+-. 0.157 100 (VC) 0.16799 0.168 1.50 0.16772 0.168 1.50
0.16085 0.161 1.44 0.25 0.06733 0.0673 0.0813 .+-. 0.0137 0.601
0.726 .+-. 0.122 51.8 0.08193 0.0819 0.732 0.09465 0.0947 0.845 2.5
0.05931 0.0593 0.0718 .+-. 0.0110 0.530 0.641 .+-. 0.0983 45.8
0.07618 0.0762 0.680 0.08000 0.0800 0.714 25 0.06804 0.0680 0.0723
.+-. 0.00488 0.608 0.645 .+-. 0.0436 46.1 0.07118 0.0712 0.636
0.07762 0.0776 0.693 Human Donor 2 0 0.02947 0.0295 0.0342 .+-.
0.00673 0.263 0.305 .+-. 0.0601 100 (VC) N/A* N/A* N/A* 0.03112
0.0311 0.278 0.04186 0.0419 0.374 0.25 0.01570 0.0157 0.0138 .+-.
0.00170 0.140 0.123 .+-. 0.0152 40.4 0.01323 0.0132 0.118 0.01243
0.0124 0.111 2.5 0.01871 0.0187 0.0191 .+-. 0.00329 0.167 0.171
.+-. 0.0293 55.9 0.01602 0.0160 0.143 0.02256 0.0226 0.201 25
0.01745 0.0175 0.0163 .+-. 0.00102 0.156 0.145 .+-. 0.00906 47.7
0.01558 0.0156 0.139 0.01583 0.0158 0.141 Human Donor 3 0 0.15807
0.158 0.162 .+-. 0.0228 1.41 1.45 .+-. 0.204 100 (VC) 0.14713 0.147
1.31 0.19520 0.195 1.74 0.14706 0.147 1.31 0.25 0.08707 0.0871
0.0871 .+-. 0.00248 0.777 0.778 .+-. 0.0221 53.8 0.08966 0.0897
0.801 0.08471 0.0847 0.756 2.5 0.08908 0.0891 0.0886 .+-. 0.00565
0.795 0.791 .+-. 0.0504 54.7 0.08271 0.0827 0.738 0.09397 0.0940
0.839 25 0.08904 0.0890 0.0947 .+-. 0.00816 0.795 0.846 .+-. 0.0729
58.5 0.10407 0.104 0.929 0.09104 0.0910 0.813 Abbreviations: SD,
standard deviation; VC, vehicle control (1% water/1% acetonitrile);
N/A, not applicable *Sample was unavailable for analysis due to
autosampler error. Note: For all calculations above, the resulting
values are shown with at least three significant figures for
display purposes only.
TABLE-US-00020 TABLE 17 CYP2C19 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition 4'-Hydroxymephenytoin formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/million
cells) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC Human Donor 1 0 0.00757.sup.a <0.0500
<0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000 100 (VC)
0.00687.sup.a <0.0500 <0.446 0.00763.sup.a <0.0500
<0.446 0.00755.sup.a <0.0500 <0.446 0.25 0.00555.sup.a
<0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000
100 0.00523.sup.a <0.0500 <0.446 (71.7%) 0.00516.sup.a
<0.0500 <0.446 2.5 0.00491.sup.a <0.0500 <0.0500 .+-.
0.0000 <0.446 <0.446 .+-. 0.000 100 0.00524.sup.a <0.0500
<0.446 (69.2%) 0.00522.sup.a <0.0500 <0.446 25
0.00457.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446
.+-. 0.000 100 0.00461.sup.a <0.0500 <0.446 (62.3%)
0.00466.sup.a <0.0500 <0.446 Human Donor 2 0 0.00871.sup.a
<0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000
100 (VC) 0.00877.sup.a <0.0500 <0.446 0.00826.sup.a
<0.0500 <0.446 0.00922.sup.a <0.0500 <0.446 0.25
0.00726.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446
.+-. 0.000 100 0.00768.sup.a <0.0500 <0.446 (82.5%)
0.00670.sup.a <0.0500 <0.446 2.5 0.00707.sup.a <0.0500
<0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000 100
0.00649.sup.a <0.0500 <0.446 (79.8%) 0.00736.sup.a <0.0500
<0.446 25 0.00704.sup.a <0.0500 <0.0500 .+-. 0.0000
<0.446 <0.446 .+-. 0.000 100 0.00612.sup.a <0.0500
<0.446 (74.5%) 0.00636.sup.a <0.0500 <0.446 Human Donor 3
0 0.01106.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446
<0.446 .+-. 0.000 100 (VC) 0.01082.sup.a <0.0500 <0.446
0.01317.sup.a <0.0500 <0.446 0.01098.sup.a <0.0500
<0.446 0.25 0.00771.sup.a <0.0500 <0.0500 .+-. 0.0000
<0.446 <0.446 .+-. 0.000 100 0.00668.sup.a <0.0500
<0.446 (64.7%) 0.00795.sup.a <0.0500 <0.446 2.5
0.00844.sup.a <0.0500 <0.0500 .+-. 0.0000 <0.446 <0.446
.+-. 0.000 100 0.00808.sup.a <0.0500 <0.446 (70.6%)
0.00786.sup.a <0.0500 <0.446 25 0.00666.sup.a <0.0500
<0.0500 .+-. 0.0000 <0.446 <0.446 .+-. 0.000 100
0.00846.sup.a <0.0500 <0.446 (65.3%) 0.00744.sup.a <0.0500
<0.446 Abbreviations: SD, standard deviation; VC, vehicle
control (1% water/1% acetonitrile) .sup.aThe observed analyzed
value (.mu.M) was below the lowest concentration on the standard
curve (0.05 .mu.M). Note: For all calculations above, the resulting
values are shown with at least three significant figures for
display purposes only.
TABLE-US-00021 TABLE 18 CYP2D6 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition Dextrorphan formation Specific Activity
Colchicine Raw Adjusted (.mu.M) (pmol/min/million cells) Percent
(.mu.M) (.mu.M) Individual Mean .+-. SD Individual Mean .+-. SD of
VC Human Donor 1 0 0.06354 0.0635 0.0652 .+-. 0.00181 0.567 0.582
.+-. 0.0161 100 (VC) 0.06416 0.0642 0.573 0.06560 0.0656 0.586
0.06761 0.0676 0.604 0.25 0.02619 0.0262 0.0285 .+-. 0.00204 0.234
0.254 .+-. 0.0182 43.6 0.02910 0.0291 0.260 0.03012 0.0301 0.269
2.5 0.02623 0.0262 0.0286 .+-. 0.00203 0.234 0.255 .+-. 0.0181 43.8
0.02949 0.0295 0.263 0.02995 0.0300 0.267 25 0.02551 0.0255 0.0288
.+-. 0.00291 0.228 0.258 .+-. 0.0260 44.2 0.03019 0.0302 0.270
0.03084 0.0308 0.275 Human Donor 2 0 0.00900.sup.a <0.0100
<0.0103 .+-. 0.000620 <0.0893 <0.0921 .+-. 0.00554 100
(VC) 0.00943.sup.a <0.0100 <0.0893 0.01124 0.0112 0.100
0.00959.sup.a <0.0100 <0.0893 0.25 0.00000.sup.a <0.0100
<0.0100 .+-. 0.000 <0.0893 <0.0893 .+-. 0.000 97.0
0.00000.sup.a <0.0100 <0.0893 0.00000.sup.a <0.0100
<0.0893 2.5 0.00000.sup.a <0.0100 <0.0100 .+-. 0.000
<0.0893 <0.0893 .+-. 0.000 97.0 0.00000.sup.a <0.0100
<0.0893 0.00000.sup.a <0.0100 <0.0893 25 0.00000.sup.a
<0.0100 <0.0100 .+-. 0.000 <0.0893 <0.0893 .+-. 0.000
97.0 0.00000.sup.a <0.0100 <0.0893 0.00000.sup.a <0.0100
<0.0893 Human Donor 3 0 0.16083 0.161 0.161 .+-. 0.00194 1.44
1.44 .+-. 0.0173 100 (VC) 0.16420 0.164 1.47 0.15972 0.160 1.43
0.16086 0.161 1.44 0.25 0.08660 0.0866 0.0842 .+-. 0.00220 0.773
0.752 .+-. 0.0196 52.2 0.08228 0.0823 0.735 0.08371 0.0837 0.747
2.5 0.08375 0.0838 0.0849 .+-. 0.00101 0.748 0.758 .+-. 0.00904
52.6 0.08517 0.0852 0.760 0.08571 0.0857 0.765 25 0.09870 0.0987
0.0932 .+-. 0.00519 0.881 0.832 .+-. 0.0464 57.8 0.08837 0.0884
0.789 0.09259 0.0926 0.827 Abbreviations: SD, standard deviation;
VC, vehicle control (1% water/1% acetonitrile) .sup.aThe observed
analyzed value (.mu.M) was below the lowest concentration on the
standard curve (0.01 .mu.M). Note: For all calculations above, the
resulting values are shown with at least three significant figures
for display purposes only.
TABLE-US-00022 TABLE 19 CYP2E1 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition 6-Hydroxychlorzoxazone formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/million
cells) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC Human Donor 1 0 0.15265 0.153 0.163 .+-. 0.0106
1.36 1.45 .+-. 0.0949 100 (VC) 0.15873 0.159 1.42 0.16111 0.161
1.44 0.17752 0.178 1.59 0.25 0.10828 0.108 0.109 .+-. 0.00240 0.967
0.977 .+-. 0.0214 67.4 0.11224 0.112 1.00 0.10791 0.108 0.963 2.5
0.11581 0.116 0.113 .+-. 0.00232 1.03 1.01 .+-. 0.0207 69.7 0.11131
0.111 0.994 0.11261 0.113 1.01 25 0.11066 0.111 0.112 .+-. 0.00738
0.988 0.997 .+-. 0.0659 68.7 0.10490 0.105 0.937 0.11955 0.120 1.07
Human Donor 2 0 0.07977 0.0798 0.0806 .+-. 0.00616 0.712 0.720 .+-.
0.0550 100 (VC) 0.08298 0.0830 0.741 0.07255 0.0726 0.648 0.08712
0.0871 0.778 0.25 0.04351 0.0435 0.0500 .+-. 0.00580 0.388 0.447
.+-. 0.0518 62.1 0.05192 0.0519 0.464 0.05464 0.0546 0.488 2.5
0.06000 0.0600 0.0588 .+-. 0.00184 0.536 0.525 .+-. 0.0164 72.9
0.05666 0.0567 0.506 0.05965 0.0597 0.533 25 0.05587 0.0559 0.0566
.+-. 0.000955 0.499 0.505 .+-. 0.00853 70.2 0.05616 0.0562 0.501
0.05765 0.0577 0.515 Human Donor 3 0 0.04012 0.0401 0.0393 .+-.
0.00443 0.358 0.351 .+-. 0.0396 100 (VC) 0.04016 0.0402 0.359
0.03314 0.0331 0.296 0.04373 0.0437 0.390 0.25 0.02186 0.0219
0.0177 .+-. 0.00512 0.195 0.158 .+-. 0.0457 45.1 0.01931 0.0193
0.172 0.01199 0.0120 0.107 2.5 0.01799 0.0180 0.0175 .+-. 0.000956
0.161 0.156 .+-. 0.00853 44.4 0.01635 0.0164 0.146 0.01802 0.0180
0.161 25 0.01562 0.0156 0.0187 .+-. 0.00311 0.139 0.167 .+-. 0.0277
47.7 0.02183 0.0218 0.195 0.01872 0.0187 0.167 Abbreviations: SD,
standard deviation; VC, vehicle control (1% water/1% acetonitrile)
Note: For all calculations above, the resulting values are shown
with at least three significant figures for display purposes
only.
TABLE-US-00023 TABLE 20 CYP3A4 Activity in Cryopreserved Human
Hepatocyte Monolayers Following 48 hr Incubation with Colchicine
Prior to Substrate Addition 6.beta.-Hydroxytestosterone formation
Specific Activity Colchicine Raw Adjusted (.mu.M) (pmol/min/million
cells) Percent (.mu.M) (.mu.M) Individual Mean .+-. SD Individual
Mean .+-. SD of VC Human Donor 1 0 0.41879 0.419 0.379 .+-. 0.0415
3.74 3.38 .+-. 0.370 100 (VC) 0.37573 0.376 3.35 0.39822 0.398 3.56
0.32251 0.323 2.88 0.25 0.11124 0.111 0.166 .+-. 0.0510 0.993 1.48
.+-. 0.456 43.8 0.21218 0.212 1.89 0.17479 0.175 1.56 2.5 0.14595
0.146 0.152 .+-. 0.00674 1.30 1.36 .+-. 0.0602 40.2 0.15160 0.152
1.35 0.15937 0.159 1.42 25* 0.00000.sup.a <0.100 <0.100 .+-.
0.000 <0.893 <0.893 .+-. 0.000 26.4 0.00000.sup.a <0.100
<0.893 0.00000.sup.a <0.100 <0.893 Human Donor 2 0
0.04579.sup.a <0.100 <0.125 .+-. 0.0172 <0.893 <1.12
.+-. 0.153 100 (VC) 0.13621 0.136 1.22 0.13055 0.131 1.17 0.13513
0.135 1.21 0.25 0.04686.sup.a <0.100 <0.100 .+-. 0.000
<0.893 <0.893 .+-. 0.000 79.7 0.08440.sup.a <0.100
<0.893 0.08079.sup.a <0.100 <0.893 2.5 0.06037.sup.a
<0.100 <0.100 .+-. 0.000 <0.893 <0.893 .+-. 0.000 79.7
0.06539.sup.a <0.100 <0.893 0.06427.sup.a <0.100 <0.893
25* 0.00000.sup.a <0.100 <0.100 .+-. 0.000 <0.893
<0.893 .+-. 0.000 79.7 0.00000.sup.a <0.100 <0.893
0.00000.sup.a <0.100 <0.893 Human Donor 3 0 0.50361 0.504
0.518 .+-. 0.0127 4.50 4.63 .+-. 0.113 100 (VC) 0.53433 0.534 4.77
0.51554 0.516 4.60 0.51948 0.519 4.64 0.25 0.22105 0.221 0.201 .+-.
0.0380 1.97 1.80 .+-. 0.339 38.9 0.22571 0.226 2.02 0.15772 0.158
1.41 2.5 0.24948 0.249 0.244 .+-. 0.00500 2.23 2.18 .+-. 0.0446
47.0 0.24106 0.241 2.15 0.24061 0.241 2.15 25* 0.00000.sup.a
<0.100 <0.118 .+-. 0.0157 <0.893 <1.05 .+-. 0.141
<22.7 0.13019 0.130 1.16 0.12285 0.123 1.10 Abbreviations: SD,
standard deviation; VC, vehicle control (1% water/1% acetonitrile)
*Test Article interference was observed in each of these samples.
.sup.aThe observed analyzed value (.mu.M) was below the lowest
concentration on the standard curve (0.1 .mu.M). Note: For all
calculations above, the resulting values are shown with at least
three significant figures for display purposes only.
[0198] CYP1A2 activity in cryopreserved human hepatocytes was
quantified by measuring the formation of acetaminophen from
phenacetin. Following treatment with 50 .mu.M omeprazole, a known
inducer for CYP1A2, CYP1A2 activity was 653%, 765%, and 596% of the
vehicle control (VC, 1% acetonitrile with 1% water) in human
hepatocytes prepared from Donors 1, 2, and 3, respectively. CYP3A4
activity in cryopreserved human hepatocytes was quantified by
measuring the formation of 6b hydroxytestosterone from
testosterone. Following treatment with 25 .mu.M rifampin, a known
inducer for CYP3A4, CYP3A4 activity was 2,796%, >2,092%, and
2,633% of the VC in human hepatocytes prepared from Donors 1, 2,
and 3, respectively. The increase in activities of CYP1A2 and
CYP3A4 following treatment with the known inducers indicate that
the hepatocytes from these donors were inducible.
[0199] Colchicine at the tested concentrations did not induce
CYP1A2 activity in human hepatocytes prepared from all three
donors, instead a significant suppression of enzyme activity was
observed. This conclusion was based on CYP1A2 activity (22.9, 26.0,
and 23.5% of the VC in Donor 1; 19.6, 20.7, and 17.9% of the VC in
Donor 2; and 23.8, 23.8, and 20.7% of the VC in Donor 3) in
hepatocytes treated with 0.25, 2.5, and 25 .mu.M colchicine (Table
12). The assay method detected no chromatographic interference from
colchicine or its metabolite.
[0200] CYP2A6 activity in cryopreserved human hepatocytes was
quantified by adding coumarin to the hepatocytes and measuring the
formation of 7-hydroxycoumarin (7-HC) and its conjugated
derivatives, 7-hydroxycoumarin glucuronide (7-HCG) and
7-hydroxycoumarin sulfate (7-HCS). Colchicine at the tested
concentrations did not induce CYP2A6 activity in human hepatocytes
prepared from all three donors, instead a significant suppression
of enzyme activity was observed. This conclusion was based on the
amount of 7-HC, 7-HCG, 7-HCS, or the sum of the above three
metabolites formed in hepatocytes treated with 0.25, 2.5, and 25
.mu.M colchicine (Tables 13a-13d). The assay method detected no
chromatographic interference from colchicine or its metabolite.
[0201] CYP2B6 activity in cryopreserved human hepatocytes was
quantified by adding S-mephenyloin to the hepatocytes and measuring
the formation of its metabolite, nirvanol. Colchicine at the tested
concentrations did not induce CYP2B6 activity in human hepatocytes
prepared from all three donors, instead significant suppression of
enzyme activity was observed. This conclusion was based on CYP2B6
activity (49.2, 48.4, and 48.3% of the VC in Donor 1; 49.3, 50.1,
and 60.0% of the VC in Donor 2; and 100, 100, and 100% of the VC in
Donor 3) in hepatocytes treated with 0.25, 2.5, and 25 .mu.M
colchicine (Table 14). The assay method detected no chromatographic
interference from colchicine or its metabolite.
[0202] CYP2C8 activity in cryopreserved human hepatocytes was
quantified by adding paclitaxel to the hepatocytes and measuring
the formation of its metabolite, 6-hydropaclitaxel. Colchicine at
the tested concentrations did not induce CYP2C8 activity in human
hepatocytes isolated from all three donors, instead significant
suppression of enzyme activity was observed. This conclusion was
based on CYP2C8 activity in hepatocytes treated with 0.25, 2.5, and
25 .mu.M colchicine (Table 15) calculated for Donors 1 and 3 using
the measured amounts of 6-hydroxypaclitaxel formed, even though
these amounts were generally less than the LLOQ for
6-hydroxypaclitaxel, and on the observation that for Donor 2 that
the measured amount of 6-hydroxypaclitaxel for the VC was lowered
to an undetectable amount in each of the samples with colchicine.
The assay method detected no chromatographic interference from
colchicine or its metabolite.
[0203] CYP2C9 activity in cryopreserved human hepatocytes was
quantified by adding tolbutamide to the hepatocytes and measuring
the formation of its metabolite, 4'-methylhydroxytolbutamide.
Colchicine at the tested concentrations did not induce CYP2C9
activity in human hepatocytes prepared from all three donors, but
did significantly suppress CYP2C9 enzyme activity. This conclusion
was based on CYP2C9 activity (51.8, 45.8, and 46.1% of the VC in
Donor 1; 40.4, 55.9, and 47.7% of the VC in Donor 2; and 53.8,
54.7, and 58.5% of the VC in Donor 3) in hepatocytes treated with
0.25, 2.5, and 25 .mu.M colchicine (Table 16). The assay method
detected no chromatographic interference from colchicine or its
metabolite.
[0204] CYP2C19 activity in cryopreserved human hepatocytes was
quantified by adding S-mephenyloin to the hepatocytes and measuring
the formation of its metabolite, 4'-hydroxymephenyloin. Levels of
4'-hydroxymephenyloin from CYP2C19 activity in hepatocytes treated
with vehicle or 0.25, 2.5 and 25 mM of colchicine was below the
LLOQ (Table 17), however when these measured values were used
colchicine at the concentrations tested did not induce CYP2C19
activity in human hepatocytes isolated from all three donors.
Instead statistically significant suppression of CYP2C19 enzyme
activity was observed in all three donors. The assay method
detected no chromatographic interference from colchicine or its
metabolite.
[0205] CYP2D6 activity in cryopreserved human hepatocytes was
quantified by adding dextromethorphan to the hepatocytes and
measuring the formation of its metabolite, dextrorphan. Colchicine
at the concentrations tested did not induce CYP2D6 activity in
human hepatocytes isolated from all three donors, but it did
suppress CYP2D6 activity at a statistically significant level. This
conclusion was based on CYP2D6 activity (43.6, 43.8, and 44.2% of
the VC in Donor 1; and 52.2, 52.6, and 57.8% of the VC in Donor 3)
in hepatocytes treated with 0.25, 2.5, and 25 .mu.M colchicine
(Table 18). For Donor 2, the VC samples were below the LLOQ for
dextrorphan, however for the samples of each of the three
colchicine concentrations tested, no dextrorphan was measurable in
the samples, an observation qualitatively consistent with
suppression of CYP2D6 activity in Donor 2 as well. The assay method
detected no chromatographic interference from colchicine or its
metabolite.
[0206] CYP2E1 activity in cryopreserved human hepatocytes was
quantified by adding chlorzoxazone to the hepatocytes and measuring
the formation of its metabolite, 6-hydroxychlorzoxazone. Colchicine
at the concentrations tested did not induce CYP2E1 activity in
human hepatocytes isolated from all three donors, but it did
suppress CYP2E1 activity at a statistically significant level. This
conclusion was based on CYP2E1 activity (67.4, 69.7, and 68.7% of
the VC in Donor 1; 62.1, 72.9, and 70.2% of the VC in Donor 2; and
45.1, 44.4, and 47.7% of the VC in Donor 3) in hepatocytes treated
with 0.25, 2.5, and 25 .mu.M colchicine (Table 19). The assay
method detected no chromatographic interference from colchicine or
its metabolite
[0207] CYP3A4 activity in cryopreserved human hepatocytes was
quantified by adding testosterone to the hepatocytes and measuring
the formation of its metabolite, 61 hydroxytestosterone. The assay
method detected chromatographic interference from colchicine or its
metabolite since colchicine or its metabolite eluted at a retention
time close to that for 61 hydroxytestosterone (data not shown). In
spite of this interference, the analytical method was still able to
quantitate the amount of 61 hydroxytestosterone formed following
treatment with colchicine at the concentrations of 0.25 or 2.5
.mu.M, but not at the highest concentration used 25 .mu.M. Although
the amount of 61 hydroxytestosterone formed following treatment
with 25 .mu.M of colchicine could not be quantitated due to this
interference, it was judged qualitatively to be less than that
following treatment with 0.25 or 2.5 .mu.M of colchicine based on
the chromatograms. Therefore, no analytical method development was
recommended and it was concluded that colchicine at the
concentrations tested did not induce CYP3A4 activity in human
hepatocytes isolated from all three donors based on the data in
Table 20, but instead suppressed CYP3A4 activity.
[0208] In summary, colchicine at the tested concentrations did not
induce activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in hepatocytes from any of the
three donors. To the contrary, colchicine actually decreased the
enzyme activity levels of the nine CYPs studied.
Example 3
Comparison of CYP1A2 Induction/Suppression Potential of Colchicine
and Vinblastine in Primary Human Hepatocytes
[0209] The purpose of this study was to assess the induction and/or
suppression of hepatic cytochrome P450 1A2 activity and mRNA
expression by colchicine and vinblastine in primary human
hepatocyte cultures prepared from three independent human
donors.
[0210] Primary cultures of human hepatocytes were prepared using
liver tissue from 3 human donors (Hu4021, Hu485, and Hu503; Table
1). Information on the liver tissue donors is shown below in Table
21.
TABLE-US-00024 TABLE 21 Liver Source Information Donor Weight
Alcohol Drug Viability after ID Sex Race Age Obese Height (lbs)
Smoking Abuse Abuse Isolation Hu485 Male Caucasian 70 No 5'9'' 190
Yes (55 yrs) No No 85% Hu4021 Female Caucasian 64 No 5'4'' 132 No
No No 84% Hu503 Male Caucasian 64 No 5'7'' 188 No No No 90%
[0211] Hepatocytes were isolated by a collagenase perfusion method
(LeCluyse, E. L., et al. (2005) Methods Mol Biol 290, 207-229).
After isolation, hepatocytes were re-suspended in Dulbecco's
modified Eagle medium (DMEM; Gibco BRL, Grand Island, N.Y.)
containing 5% fetal bovine serum (FBS; Gibco BRL), insulin and
dexamethasone (Gibco BRL) and added to plates coated with a
collagen type I substratum (BD Biosciences, Bedford, Mass.). After
attachment, serum-free William's E medium containing dexamethasone,
insulin, transferrin, selenium (ITS.sup.+, BD Biosciences) was
added. Cultures of hepatocytes were maintained for 36 to 48 hours
prior to initiating experiments.
[0212] Hepatocyte cultures were treated for 3 consecutive days with
colchicine (0.22, 2.2, and 22 .quadrature.M; Sanmar Specialty
Chemicals, Ltd., Chemai, India), vinblastine (1, 10, and 100 nM;
Sigma Chemical Co, St. Louis, Mo.) and the positive control CYP1A
inducer 3-methylcholanthrene (3-MC; 2 .quadrature.M; Sigma Chemical
Co.). Dosing solutions were prepared fresh daily in cell culture
medium such that the final dimethyl sulfoxide (DMSO) concentration
was 0.1%. At the conclusion of the treatment period, cells were
incubated with substrates and harvested for total RNA
isolations.
[0213] After completion of the treatment period, medium was
aspirated, the cells were rinsed once with fresh medium, and
William's E containing 100 .mu.M phenacetin, a CYP450 marker
substrate for CYP1A2, was added directly to the monolayers. Plates
were incubated at 37.degree. C. in a humidified chamber while
mixing on an orbital shaker for 30 minutes. At the end of the
incubation period, medium samples were collected and analyzed.
Analysis of the in situ CYP1A2-mediated metabolism of phenacetin to
APAP was performed by liquid chromatography coupled with mass
spectrometry (LC-MS/MS). Mass spectrometric data were acquired,
integrated, regressed, and quantified with MASSLYNX software,
version 3.4 (Micromass, Manchester, UK).
[0214] Cells in 24-well format were washed with one volume (0.5 mL)
of Hank's Balanced Salt Solution and lysed by addition of 350 .mu.L
of ABI Nucleic Acid Lysis Solution (Applied Biosystems, Foster
City, Calif.) and frozen at -70.degree. C. Lysates were thawed and
total RNA was isolated from each sample using an ABI 6100
Prepstation. Isolated RNA was analyzed using a Nanoprop.RTM.
spectrophotometer (Wilmington, Del.) to estimate the total RNA
concentrations, and RNA was stored at -70.degree. C. For reverse
transcription (RT), approximately 200 ng of pooled total RNA was
converted to cDNA following the manufacturer's procedure for the
High Capacity cDNA Archive Kit (Applied Biosystems, Foster City,
Calif.).
[0215] CYP1A2 cDNA from human hepatocyte cultures was analyzed from
each RT reaction using gene-specific TAQMAN.RTM. primer/probe sets
(Applied Biosystems). Reactions with the specific primer/probes for
beta-actin were analyzed as an endogenous control for CYP450
expression. Amplifications were performed on an ABI 7500 Real-Time
PCR System (Applied Biosystems) in relative quantification mode for
40 amplification cycles using standard conditions for
TaqMan.RTM.-based assays. Threshold cycle (CT) determinations were
performed by the ABI 7500 system software for both CYP450 and
endogenous control gene .quadrature.-actin. Relative-fold mRNA
content was determined for each treatment group relative to the
endogenous control gene expression and 0.1% DMSO vehicle control
for each sample using the relationship:
2.sup.-.DELTA..DELTA.C.sup.T=relative-fold mRNA content.
[0216] Data (activities and mRNA) were analyzed for mean and
standard error. Statistical analysis of data using multiple
comparisons methods was performed with XLSTAT 2006 (Addinsoft, New
York, N.Y.) to determine which treatment groups were statistically
different from the vehicle control group. Initially, data were
examined for homoscedasticity and normality (of residuals from the
ANOVA model fitting) using Levene's and Shapiro-Wilk tests,
respectively. If either normality of residuals or homoscedasticity
did not exist at the p.ltoreq.0.01 level, then the response
variable was transformed using log transformation (natural log) to
achieve both homoscedasticity and normality. Once normality of
residuals and homoscedasticity were achieved at the p.ltoreq.0.01
level, Dunnett's t-test was used to perform multiple comparisons
analysis versus vehicle control to determine statistically
significant differences at p.ltoreq.0.05.
[0217] Treatment of each of the 9 sample groups is summarized in
Table 22 below. 3-methylcholanthrene (3-MC) at 2 .mu.M was used as
the positive induction control. Negative control cultures were
treated with media alone (in the presence or absence of 0.1%
DMSO).
TABLE-US-00025 TABLE 22 Summary of Treatment Groups GROUP #
TREATMENT 1 Negative control (Media only) 2 Negative Control:
(Media + 0.1% DMSO) 3 CYP1A Positive Control: (Media + 2 .mu.M
3-MC) 4 Colchicine (0.22 .mu.M) 5 Colchicine (2.2 .mu.M) 6
Colchicine (22 .mu.M) 7 Vinblastine (1 nM) 8 Vinblastine (10 nM) 9
Vinblastine (100 nM)
[0218] All subsequent calculations were performed at machine
precision with the software program Microsoft Excel (Redmond,
Wash.). Enzyme activity (V) was calculated as shown in Equation
1:
V = [ X ] .times. Vol M X .times. T .times. C , Equation 1
##EQU00001##
[0219] where [X] is the concentration of APAP, Vol is the volume of
the incubation, M.sub.X is the molecular weight of APAP, T is the
time of the incubation, and C is the number of cells in the
incubation volume.
[0220] To quantify the inducibility of CYP1A2, the relative fold
changes were calculated as the ratio of the mean of the activity of
the treated samples to the vehicle (0.1% DMSO) control activity, as
shown in Equation 2, where .mu. is the mean activity of the
replicate samples for the group designated within the
parentheses.
Fold Induction = .mu. ( sample ) .mu. ( DMSO ) Equation 2
##EQU00002##
[0221] The percent of treatment induction response as compared to
the positive control induction response was calculated as shown in
Equation 3.
% Positive Control Response = .mu. ( sample ) - .mu. ( DMSO ) .mu.
( positive control ) - .mu. ( DMSO ) .times. 100 , Equation 3
##EQU00003##
where .mu.(sample) is the mean of the sample activities, .mu.(DMSO)
is the mean activity of 0.1% DMSO-treated vehicle control samples,
and .mu.(positive control) is the mean activity of positive control
(3-MC)-treated samples.
[0222] Similarly, the percent suppression of enzyme activity
observed relative to the vehicle control samples was calculated
using Equation 4:
Percent Suppression = .mu. ( DMSO ) - .mu. ( sample ) .mu. ( DMSO )
.times. 100 Equation 4 ##EQU00004##
[0223] The presence or absence of acute cytotoxicity to hepatocytes
was determined by evaluating morphological changes and lactate
dehydrogenase leakage following treatment with colchicine or
vinblastine.
[0224] The morphology of the cultures was assessed on
representative treatments and compared to control cultures. Cell
integrity was evaluated using phase contrast microscopy;
morphological alterations were noted (e.g., cell shape, cytoplasmic
alterations, accumulation of vacuoles suggestive of dilated
organelles and lipid droplets). Images were captured using a Zeiss
Axiovert inverted research microscope equipped with phase-contrast
optics, a 3 CCD camera, and imaging computer with image analysis
software to document appearance observed in samples as compared to
vehicle controls. No marked changes in cell morphology consistent
with cytotoxicity were observed with cultures of hepatocytes
treated at colchicine concentrations of 0.22-22 .mu.M. Only the
highest concentration of vinblastine, 100 nM, caused any change in
cell morphology; the change observed was slight.
[0225] The inability of hepatocytes to retain intracellular enzymes
is an indicator of irreversible damage to the plasma membrane. To
determine membrane integrity, lactate dehydrogenase (LDH) leakage
from hepatocytes was measured. Lactate dehydrogenase (LDH) leakage
from hepatocytes treated with colchicine and vinblastine was
measured in the culture media and compared with that of controls at
72 hours. LDH leakage was determined with the CytoTox One LDH assay
system (Promega Corporation, Madison, Wis.) according to the
manufacturer's instructions. The activity in the medium was
expressed as percentage of the LDH activity from control cells that
were completely lysed by sonication. LDH activity in the three cell
lines at 22 .mu.M colchicine was 0.8% (HU503), 6.7% (HU485), and
7.7% (HU4021), while LDH activity in the three cell lines at 100 nM
vinblastine was 0.4% (HU503), 6.8% (HU485), and 3.6% (HU4021) and
in the vehicle control was 0.6% (HU503), 6.0% (HU485), and 3.7%
(HU4021). LDH activity observed in each of the three different cell
lines varied little with colchicine or vinblastine
concentration.
[0226] Enzymatic activity determinations for each of the nine
sample groups are shown below in Tables 23-25, for each of the
three hepatocyte donors, respectively. Levels of CYP1A2 mRNA
content determined by Quantitative RT-PCR analysis is presented for
each of the nine sample groups for each of the three hepatocyte
donors in Tables 26-28.
TABLE-US-00026 TABLE 23 Phenacetin O-Dealkylation Assay for the
Determination of CYP1A2 Activity Induction in Human Hepatocytes;
Hepatocyte Lot # Hu485 Activity Mean Activity % Conc. (pmol/min/
(pmol/min/ Standard of Fold % Positive Treatment Sample ID (ng/mL)
million cells) million cells) Error VC Change Control Hu485-0-1
29.7 8.74 Hu485-0-2 29.3 8.62 Control (No DMSO) Hu485-0-3 24.2 7.10
8.15 0.53 Hu485-1-1 19.6 5.76 Hu485-1-2 15.8 4.63 Control (0.1%
DMSO) Hu485-1-3 17.7 5.19 5.19 0.33 1.0 Hu485-2-1 2070 609
Hu485-2-2 1550 455 3-MC (2 .mu.M) Hu485-2-3 1390 408 491 60 94.5
100 Hu485-3-1 8.71 2.56 Hu485-3-2 8.16 2.40 Colchicine (0.22 .mu.M)
Hu485-3-3 6.11 1.80 2.25 0.23 43.4 0.4 -0.61 Hu485-4-1 6.59 1.94
Hu485-4-2 8.45 2.48 Colchicine (2.2 .mu.M) Hu485-4-3 8.31 2.44 2.29
0.18 44.1 0.4 -0.60 Hu485-5-1 10.1 2.96 Hu485-5-2 6.27 1.84
Colchicine (22 .mu.M) Hu485-5-3 5.95 1.75 2.18 0.39 42.0 0.4 -0.62
Hu485-6-1 41.7 12.3 Hu485-6-2 46.9 13.8 Vinblastine (1 nM)
Hu485-6-3 45.1 13.3 13.1 0.4 252 2.5 1.6 Hu485-7-1 43.1 12.7
Hu485-7-2 56.2 16.5 Vinblastine (10 nM) Hu485-7-3 42.6 12.5 13.9
1.3 268 2.7 1.8 Hu485-8-1 33.8 9.93 Hu485-8-2 32.7 9.61 Vinblastine
(100 nM) Hu485-8-3 27.7 8.15 9.23 0.55 178 1.8 0.83
TABLE-US-00027 TABLE 24 Phenacetin O-Dealkylation Assay for the
Determination of CYP1A2 Activity Induction in Human Hepatocytes;
Hepatocyte Lot # Hu4021 Activity Mean Activity % Conc. (pmol/min/
(pmol/min/ Std of Fold % Positive Treatment Sample ID (ng/mL)
million cells) million cells) Error VC Change Control Control (No
Hu4021-0-1 10.9 3.20 DMSO) Hu4021-0-2 18.9 5.55 Hu4021-0-3 16.0
4.69 Hu4021-0-4 14.2 4.17 Hu4021-0-5 12.0 3.52 Hu4021-0-6 15.1 4.44
4.26 0.35 Control (0.1% Hu4021-1-1 21.3 6.26 DMSO) Hu4021-1-2 26.3
7.72 Hu4021-1-3 21.2 6.25 Hu4021-1-4 18.1 5.32 Hu4021-1-5 23.6 6.95
Hu4021-1-6 31.0 9.11 6.93 0.54 1.0 3-MC (2 .mu.M) Hu4021-2-1 1160
341 Hu4021-2-2 1060 311 Hu4021-2-3 1170 343 Hu4021-2-4 1150 339
Hu4021-2-5 1280 376 Hu4021-2-6* N26 1740 511 342 10 49.3 100
Colchicine Hu4021-3-1 12.2 3.60 (0.22 .mu.M) Hu4021-3-2 12.4 3.63
Hu4021-3-3 8.25 2.43 3.22 0.40 46.5 0.5 -1.1 Colchicine Hu4021-4-1
6.39 1.88 (2.2 .mu.M) Hu4021-4-2 10.5 3.08 Hu4021-4-3 6.37 1.87
2.28 0.40 32.9 0.3 -1.4 Colchicine Hu4021-5-1 8.71 2.56 (22 .mu.M)
Hu4021-5-2 7.21 2.12 Hu4021-5-3 6.34 1.86 2.18 0.20 31.5 0.3 -1.4
Vinblastine Hu4021-6-1 27.2 7.99 (1 nM) Hu4021-6-2 22.0 6.47
Hu4021-6-3 24.0 7.06 7.17 0.44 103.5 1.0 0.1 Vinblastine Hu4021-7-1
65.4 19.2 (10 nM) Hu4021-7-2 61.4 18.0 Hu4021-7-3 55.1 16.2 17.8
0.9 256.9 2.6 3.2 Vinblastine Hu4021-8-1 17.7 5.20 (100 nM)
Hu4021-8-2 18.3 5.39 Hu4021-8-3 19.1 5.63 5.41 0.12 78.1 0.8 -0.5
N26 - NOT INCLUDED IN CALCULATIONS; OUTLIER BASED ON GRUBB'S
TEST.
TABLE-US-00028 TABLE 25 Phenacetin O-Dealkylation Assay for the
Determination of CYP1A2 Activity Induction in Human Hepatocytes;
Hepatocyte Lot # Hu503 Activity Mean Activity % Conc. (pmol/min/
(pmol/min/ Standard of Fold % Positive Treatment Sample ID (ng/mL)
million cells) million cells) Error VC Change Control Hu503-0-1
57.3 16.8 Hu503-0-2 59.7 17.6 Hu503-0-3 58.8 17.3 Hu503-0-4 69.2
20.3 Hu503-0-5 58.8 17.3 Control (No DMSO) Hu503-0-6 52.7 15.5 17.5
0.6 Hu503-1-1 55.5 16.3 Hu503-1-2 53.5 15.7 Hu503-1-3 62.7 18.4
Hu503-1-4 53.9 15.9 Hu503-1-5 57.0 16.7 Control (0.1% DMSO)
Hu503-1-6 58.6 17.2 16.7 0.4 1.0 Hu503-2-1 E2 2270 666 Hu503-2-2 E2
2000 589 Hu503-2-3 E2 2070 609 Hu503-2-4 1950 573 Hu503-2-5 E2 2070
608 3-MC (2 .mu.M) Hu503-2-6 E2 2270 667 619 16 37.0 100 Hu503-3-1
44.9 13.2 Hu503-3-2 39.1 11.5 Colchicine (0.22 .mu.M) Hu503-3-3
40.0 11.8 12.2 0.5 73.1 0.7 -0.76 Hu503-4-1 28.0 8.22 Hu503-4-2
34.4 10.1 Colchicine (2.2 .mu.M) Hu503-4-3 34.4 10.1 9.49 0.63 56.8
0.6 -1.2 Hu503-5-1 31.3 9.21 Hu503-5-2 29.3 8.63 Colchicine (22
.mu.M) Hu503-5-3 21.6 6.34 8.06 0.88 38.0 0.5 -1.4 Hu503-6-1 72.9
21.4 Hu503-6-2 68.0 20.0 Vinblastine (1 nM) Hu503-6-3 63.6 18.7
20.0 0.8 1.2 0.55 Hu503-7-1 68.6 20.2 Hu503-7-2 69.0 20.3
Vinblastine (10 nM) Hu503-7-3 65.3 19.2 19.9 0.3 1.2 0.53 Hu503-8-1
58.8 17.3 Hu503-8-2 55.0 16.2 Vinblastine (100 nM) Hu503-8-3 57.8
17.0 16.8 0.3 1.0 0.02 E2 - ESTIMATED VALUE; ABOVE THE ULOQ.
TABLE-US-00029 TABLE 26 Quantitative RT-PCR Analysis of CYP1A2 mRNA
Content Induction In Human Hepatocytes; Hepatocyte Lot# Hu485
Sample Relative Fold Mean Relative Fold Standard Percent of
Adjusted Treatment ID mRNA Content mRNA Content Error Positive
Control DMSO (0.1%) Hu485-1 1.00 0.817 0.14 0.00 Hu485-1 0.537
Hu485-1 0.913 3-MC (2 .mu.M) Hu485-2 361 396 25.50 100.00 Hu485-2
445 Hu485-2 380 Colchicine (0.22 .mu.M) Hu485-3 0.056 0.057 0.003
-0.19 Hu485-3 0.054 Hu485-3 0.063 Colchicine (2.2 .mu.M) Hu485-4
0.064 0.056 0.004 -0.19 Hu485-4 0.055 Hu485-4 0.050 Colchicine (22
.mu.M) Hu485-5 0.036 0.029 0.004 -0.20 Hu485-5 0.024 Hu485-5 0.029
Vinblastine (1 nM) Hu485-6 6.18 5.68 0.34 1.23 Hu485-6 5.02 Hu485-6
5.83 Vinblastine (10 nM) Hu485-7 5.88 5.47 0.40 1.18 Hu485-7 4.66
Hu485-7 5.86 Vinblastine (100 nM) Hu485-8 3.48 3.59 0.09 0.70
Hu485-8 3.52 Hu485-8 3.78
TABLE-US-00030 TABLE 27 Quantitative RT-PCR Analysis of CYP1A2 mRNA
Content Induction In Human Hepatocytes; Hepatocyte Lot# Hu4021
Relative Fold mRNA Mean Relative Fold Standard Percent of Positive
Treatment Sample ID Content mRNA Content Error Control DMSO (0.1%)
Hu4021-1 1.00 0.894 0.07 0.00 Hu4021-1 0.753 Hu4021-1 0.930 3-MC (2
.mu.M) Hu4021-2 68.7 71.9 2.97 100.00 Hu4021-2 69.1 Hu4021-2 77.8
Colchicine (0.22 .mu.M) Hu4021-3 0.071 0.047 0.01 -1.19 Hu4021-3
0.023 Hu4021-3 0.047 Colchicine (2.2 .mu.M) Hu4021-4 0.017 0.015
0.002 -1.24 Hu4021-4 0.011 Hu4021-4 0.016 Colchicine (22 .mu.M)
Hu4021-5 0.025 0.026 0.004 -1.22 Hu4021-5 0.020 Hu4021-5 0.033
Vinblastine (1 nM) Hu4021-6 0.259 0.229 0.03 -0.94 Hu4021-6 0.162
Hu4021-6 0.264 Vinblastine (10 nM) Hu4021-7 1.70 1.88 0.19 1.38
Hu4021-7 1.68 Hu4021-7 2.25 Vinblastine (100 nM) Hu4021-8 0.357
0.320 0.02 -0.81 Hu4021-8 0.280 Hu4021-8 0.323
TABLE-US-00031 TABLE 28 Quantitative RT-PCR Analysis of CYP1A2 mRNA
Content Induction In Human Hepatocytes; Hepatocyte Lot# Hu503
Relative Fold mRNA Mean Relative Fold Standard Percent of Positive
Treatment Sample ID Content mRNA Content Error Control DMSO (0.1%)
Hu503-1 1.00 0.914 0.04 0.00 Hu503-1 0.867 Hu503-1 0.875 3-MC (2
.mu.M) Hu503-2 167 161 12.74 100.00 Hu503-2 137 Hu503-2 180
Colchicine (0.22 .mu.M) Hu503-3 0.030 0.025 0.01 -0.55 Hu503-3
0.015 Hu503-3 0.031 Colchicine (2.2 .mu.M) Hu503-4 0.015 0.015
0.001 -0.56 Hu503-4 0.013 Hu503-4 0.016 Colchicine (22 .mu.M)
Hu503-5 0.016 0.018 0.001 -0.56 Hu503-5 0.018 Hu503-5 0.020
Vinblastine (1 nM) Hu503-6 4.18 3.604 0.29 1.68 Hu503-6 3.22
Hu503-6 3.40 Vinblastine (10 nM) Hu503-7 3.70 3.31 0.23 1.49
Hu503-7 3.32 Hu503-7 2.90 Vinblastine (100 nM) Hu503-8 1.01 0.896
0.06 -0.01 Hu503-8 0.816 Hu503-8 0.865
[0227] A marked induction of CYP1A2-catalyzed APAP formation from
phenacetin was observed with the positive control 3-MC (varying
from 37.1 to 94.6-fold induction relative to the activity of the
vehicle control (medium+0.1% DMSO) for the three hepatocyte
preparations), demonstrating that the hepatocyte cultures were
responding appropriately to a prototypical CYP1A inducer (Tables
25-27). At the concentrations tested, no induction of CYP1A
activity was observed in human hepatocytes treated with colchicine
at any of the concentrations examined, consistent with the data in
Examples 1 and 2. Instead, a marked suppression of CYP1A2 enzyme
activity was observed in all three preparations of human hepatocyte
cultures, as observed in the Example 2 experiment shown in Table
13. In the current experiments, suppression ranged from 27.3% to
68.5% lower than the vehicle control for CYP1A2 enzyme activity
(Tables 25-27). Observed CYP1A2 activity as a percent of the VC
activity after treatment with 0.22, 2.2, and 22 .mu.M colchicine,
respectively was 43.4%, 44.1%, and 42.0% for Donor Hu485; 46.5%,
32.9%, and 31.5% for Donor Hu4021; and 73.1%, 56.8%, and 38.0% for
Donor Hu503. These substantial reductions from the VC in hepatocyte
cultures from three different human donors confirm the suppression
of CYP1A2 enzyme activity observed in Donors 1-3 (Table 13) in
Example 2. In contrast, for vinblastine, the alternate tubulin
network disrupting agent tested, a very small induction (relative
to the positive control response) was observed that ranged from
-0.46 to 3.2% of the fold-change of the positive control.
[0228] A marked induction of CYP1A2 mRNA content was observed with
the positive control inducer, 3-MC, over the vehicle control. The
mean relative fold mRNA content ranged from 71.9 to 396 for the
three hepatocyte preparations (Tables 28-30), demonstrating that
the hepatocyte cultures were responding appropriately to the
prototypical CYP1A inducer. No induction of CYP1A2 mRNA content was
observed with colchicine treatment at any concentration (Tables
28-30), consistent with the observed lack of induction of enzyme
activity by colchicine. Instead, a marked suppression of mRNA
content was observed in all three preparations of human hepatocyte
cultures. The mean relative fold mRNA content observed after
treatment with colchicine at 0.22, 2.2, and 22 .mu.M was only
0.057, 0.056, and 0.029 for Donor Hu485 (Table 28); 0.047, 0.015,
and 0.026 for Donor Hu4021 (Table 29); and 0.025, 0.015, and 0.018
for Donor Hu503 (Table 30).
[0229] In contrast, for vinblastine, no marked suppression of
enzyme activity or mRNA expression was observed with vinblastine at
the concentrations examined. Instead, either a slight suppression
or a small induction (with both suppression and induction expressed
relative to the positive control induction) was observed in all
three donor preparations of primary human hepatocyte cultures. The
observed effect ranged from -0.94% to 1.68% of the positive control
response.
[0230] Therefore, colchicine has a concentration-dependent
suppressive effect on CYP1A2 mRNA expression and CYP1A2 enzyme
activity at concentrations from 0.22 to 22 .mu.M, while
vinblastine, another tubulin disrupting compound, does not suppress
CYP1A2 expression. Cytotoxicity did not appear to be the cause of
the suppression observed at the range of colchicine concentrations
studied.
Example 4
Metabolic Phenotyping of Colchicine
[0231] The purpose of this study was to identify the cytochrome
P450 enzymes involved in the in vitro metabolism of Colchicine.
[0232] Human liver microsomes pooled from 15 individuals (male and
female) (Pool HMMC-PL020; CellzDirect, Inc., In Vitro Products and
Services Division) were utilized in some experiments of this study.
This pool of human liver microsomes was characterized by
CellzDirect, Inc. with respect to donor medical history, major
cytochrome P450 enzyme activities and kinetic parameters, as well
as for polymorphic forms for CYP2C9, CYP2C19, and CYP2D6 present in
the individual donors of the pool.
[0233] Colchicine (MW=399.43, Lot # COL0206002 purity=97.05%) stock
solutions at 430 .mu.M were prepared in methanol and stored at
-20.degree. C. Stocks were diluted daily in the appropriate buffers
such that the final organic solvent concentration was <1%.
[0234] Potassium phosphate monobasic, potassium phosphate dibasic,
NADPH tetrasodium salts, and other reagents were purchased from
Sigma Chemical Co. or equivalent vendors. Methanol (HPLC grade),
water (HPLC grade), ethyl acetate, and other solvents were
purchased from Fisher, Burdick & Jackson, J. T. Baker,
Mallinckrodt, or equivalent vendors. All inhibitors were of the
highest purity available. Individual suppliers are as follows:
furafylline, pilocarpine, thio-TEPA, quercetin, sulfaphenazole,
ticlopidine, quinidine, 4 methylpyrazole, and ketoconazole were
obtained from Sigma Chemical Co.
[0235] A validated isocratic LC-MS/MS was developed to allow for
chromatographic resolution and quantitation of colchicine contained
within an incubation matrix. The following LC-MS/MS method was used
for Colchicine quantitation:
TABLE-US-00032 TABLE 29 LC/MS method for colchicine quantitation.
Substrate: Metabolite: Colchicine Standard Metabolite Range: 4.31
to 552 .mu.M Mobile Phase (isocratic): 50% Methanol, 50% 1 mM
Ammonium Acetate Buffer, 0.1% Trifluroacetic Acid (TFA) Detection
Method: LC-MS/MS HPLC Column: AQ12 Flow rate (approx.): 0.3 mL/min
Source: Electrospray (positive ion) Run Time (approx.): 2 minutes
MRM (Colchicine): 400 .fwdarw. 310 Quantitation: Least Squares
Regression 1/X Weighting
[0236] For this example, Micromass MASSLYNX.RTM. software (version
3.4, Manchester, UK) was used to collect and process
chromatographic data. Data were graphed with the software program
Microsoft EXCE.RTM. 2003 (Redmond, Wash.). Percent turnover of
colchicine was calculated using Microsoft EXCEL at machine
precision, using the following equation:
% Turnover=100-{[TA(final)]/[mean of TA(0 min)]}.times.100,
where TA(sample group)=Test Article (i.e., colchicine) and the
particular sample group is noted within the parentheses.
[0237] Incubations of colchicine with pooled human liver microsomes
were performed to establish appropriate protein concentrations and
time points for Colchicine turnover. In particular, colchicine at
43 and 430 nM was incubated with 0, 0.02, 0.05, 0.1, 0.25 and 0.5
mg/mL pooled human liver microsomal protein for 30 minutes at
37.degree. C. In addition, 43 and 430 nM of colchicine was
incubated with 0.1 mg/mL pooled human liver microsomal protein for
0, 5, 10, 20, 40, and 60 minutes at 37.degree. C. Incubation
mixtures were prepared in 0.1 M phosphate buffer, pH 7.4. The
reactions were initiated by addition of 1 mM NADPH. Reactions were
performed in triplicate and were terminated at the appropriate time
points by addition of 1 volume (relative to total reaction volume)
of methanol. Negative controls (either no NADPH or heat-treated
microsomes) were included to account for any non-enzymatic
dependent reactions. The samples were centrifuged at approximately
3000 rpm and the clear supernatant was transferred to a clean tube
and analyzed by the LC-MS/MS method described above.
[0238] Incubations with 43 and 430 nM colchicine with 0.1 mg/mL of
pooled human liver microsomal protein for 0, 5, 10, 20, 40 and 60
minutes resulted in a decrease in colchicine concentration of less
than 5% after 60 minutes incubations. These changes in colchicine
concentration were not consistently greater in magnitude than the
sample-to-sample variability.
[0239] Based on this matrix of data, a pooled human liver
microsomal protein concentration of 1 mg/mL, and an incubation time
of 60 minutes were used in subsequent experiments using the
microsomal system.
[0240] Experiments were then performed to identify CYP450
isoform(s) involved in the metabolism of colchicine in a microsomal
system using selective CYP chemical inhibitors. The microsomal
system approximates the in vivo distribution of hepatic
enzymes.
[0241] Isoform-selective chemical inhibitors were used to evaluate
the effects of individual CYP450s in human liver microsomes on the
metabolism of colchicine. The isoform-selective chemical inhibitors
and concentrations used are detailed in Table 30 below.
TABLE-US-00033 TABLE 30 Isoform-selective chemical inhibitors and
concentrations P450 Isoform Selective chemical inhibitor
Concentration CYP1A2 Furafylline 50 .mu.M CYP2A6 Pilocarpine 100
.mu.M CYP2B6 Thio-TEPA 75 .mu.M CYP2C8 Quercetin 10 .mu.M CYP2C9
Sulfaphenazole 20 .mu.M CYP2C19 Ticlopidine 1 .mu.M CYP2D6
Quinidine 10 .mu.M CYP2E1 4-Methylpyrazole 500 .mu.M CYP3A4
Ketoconazole 1 .mu.M
[0242] Results are provided in Tables 31 and 32. Colchicine was
only weakly metabolized by NADPH-dependent CYP450s with
approximately less than 5% disappearance at the conditions
examined. As turnover of colchicine in microsomes was found to be
below the experimental noise, determinations of percent inhibition
of turnover were not determined in these experiments.
TABLE-US-00034 TABLE 31 Chemical Inhibitor Data in Human Liver
Microsomes with 43 nM Colchicine Conc. Mean % Turnover Inhibitor
CYP Sample ID (nM) Conc. (nM) % Turnover Mean Std. Error 50
nM-T0-P1.0-1 43.8 50 nM-T0-P1.0-2 47.4 0 min. 50 nM-T0-P1.0-3 45.8
45.7 50 nM-T60-P1.0-1 37.1 18.7 50 nM-T60-P1.0-2 45.5 0.30 60 min.
50 nM-T60-P1.0-3 48.7 43.8 -6.5 4.2 7.6 50 nM-T60-P1.0-F-1 42.8 6.2
50 nM-T60-P1.0-F-2 48.1 -5.2 Furafylline 1A2 50 nM-T60-P1.0-F-3
45.7 45.5 0.06 0.35 3.29 50 nM-T60-P1.0-P-1 45.8 -0.26 50
nM-T60-P1.0-P-2 48.8 -6.8 Pilocarpine 2A6 50 nM-T60-P1.0-P-3 45.8
46.8 -0.29 -2.4 2.2 50 nM-T60-P1.0-TT-1 46.3 -1.4 50
nM-T60-P1.0-TT-2 47.8 -4.6 Thio-TEPA 2B6 50 nM-T60-P1.0-TT-3 50.1
48.1 -9.6 -5.2 2.4 50 nM-T60-P1.0-Qr-1 45.8 -0.31 50
nM-T60-P1.0-Qr-2 46.6 -2.0 Quercetin 2C8 50 nM-T60-P1.0-Qr-3 45.3
45.9 0.79 -0.52 0.83 50 nM-T60-P1.0-S-1 48.5 -6.1 50
nM-T60-P1.0-S-2 48.3 -5.8 Sulfaphenazole 2C9 50 nM-T60-P1.0-S-3
45.0 47.3 1.5 -3.5 2.5 50 nM-T60-P1.0-Ti-1 52.4 -14.8 50
nM-T60-P1.0-Ti-2 48.6 -6.4 Ticlopidine 2C19 50 nM-T60-P1.0-Ti-3
48.4 49.8 -5.9 -9.0 2.9 50 nM-T60-P1.0-Qi-1 50.0 -9.5 50
nM-T60-P1.0-Qi-2 47.7 -4.4 Quinidine 2D6 50 nM-T60-P1.0-Qi-3 48.6
48.8 -6.5 -6.8 1.5 50 nM-T60-P1.0-4MP-1 45.6 0.24 50
nM-T60-P1.0-4MP-2 43.4 4.9 4-Methylpyrazole 2E1 50
nM-T60-P1.0-4MP-3 43.7 44.2 4.3 3.2 1.5 50 nM-T60-P1.0-K-1 48.4
-5.9 50 nM-T60-P1.0-K-2 51.8 -13.3 Ketoconazole 3A4 50
nM-T60-P1.0-K-3 48.4 49.5 -6.0 -8.4 2.5
TABLE-US-00035 TABLE 32 Chemical Inhibitor Data in Human Liver
Microsomes with 430 nM Colchicine Conc. Mean % % Turnover Inhibitor
CYP Sample ID (nM) Conc. (nM) Turnover Mean Std. Error 500
nM-T0-P1.0-1 468 500 nM-T0-P1.0-2 463 0 min. 500 nM-T0-P1.0-3 464
465 500 nM-T60-P1.0-1 458 1.5 500 nM-T60-P1.0-2 456 1.9 60 min. 500
nM-T60-P1.0-3 441 452 5.2 2.8 1.2 500 nM-T60-P1.0-F-1 441 5.2 500
nM-T60-P1.0-F-2 457 1.6 Furafylline 1A2 500 nM-T60-P1.0-F-3 463 454
0.45 2.4 1.4 500 nM-T60-P1.0-P-1 468 -0.77 500 nM-T60-P1.0-P-2 442
4.9 Pilocarpine 2A6 500 nM-T60-P1.0-P-3 456 456 1.8 2.0 1.6 500
nM-T60-P1.0-TT-1 461 -0.77 500 nM-T60-P1.0-TT-2 466 -0.33 Thio-TEPA
2B6 500 nM-T60-P1.0-TT-3 462 463 0.54 0.33 0.34 500
nM-T60-P1.0-Qr-1 473 -1.9 500 nM-T60-P1.0-Qr-2 470 -1.1 Quercetin
2C8 500 nM-T60-P1.0-Qr-3 470 471 -1.1 -1.4 0.3 500 nM-T60-P1.0-S-1
448 3.5 500 nM-T60-P1.0-S-2 541 -16.5 Sulfaphenazole 2C9 500
nM-T60-P1.0-S-3 522 504 -12.3 -8.4 6.1 500 nM-T60-P1.0-Ti-1 493
-6.1 500 nM-T60-P1.0-Ti-2 502 -8.1 Ticlopidine 2C19 500
nM-T60-P1.0-Ti-3 N14 NA 498 NA -7.1 1.0 500 nM-T60-P1.0-Qi-1 479
-3.0 500 nM-T60-P1.0-Qi-2 469 -0.85 Quinidine 2D6 500
nM-T60-P1.0-Qi-3 474 474 -2.1 -2.0 0.6 500 nM-T60-P1.0-4MP-1 462
0.61 500 nM-T60-P1.0-4MP-2 476 -2.3 4-Methylpyrazole 2E1 500
nM-T60-P1.0-4MP-3 466 468 -0.30 -0.68 0.88 500 nM-T60-P1.0-K-1 489
-5.3 500 nM-T60-P1.0-K-2 485 -4.4 Ketoconazole 3A4 500
nM-T60-P1.0-K-3 487 487 -4.7 -4.8 0.3 N14 - NOT INCLUDED IN
CALCULATIONS; NO PEAK DETECTED.
[0243] Experiments with individual human recombinant cytochrome
P450 enzymes were conducted to determine which specific CYP450s
metabolize colchicine.
[0244] Commercially available microsomes from baculovirus-infected
insect cells containing singly-expressed recombinant human CYP
enzymes and cDNA-expressed human cytochrome p450 oxidoreductase [BD
SUPERSOMES.RTM. Enzymes; BD Biosciences Discovery Labware (Woburn,
Mass.)] were used. For CYP2A6, CYP2C9, CYP2C19, and CYP2E1, the
SUPERSOMES.RTM. also expressed human cytochrome b5 in addition to
human cytochrome p450 oxidoreductase and the human CYP isozyme. For
CYP2C9 and CYP2D6, SUPERSOMES singly-expressing different allelic
variants of the cytochrome p450 isozyme were commercially
available. For each of CYP2C9 and CYP2D6, only the *1 allele was
tested in these experiments.
[0245] Incubation mixtures containing CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4 expressed
enzymes at 5 or 20 pmol cytochrome P450 were incubated in 0.1 M
potassium phosphate buffer, pH 7.4 with .beta.-NADPH (1 mM) and
with colchicine for 60 minutes at 37.degree. C. The incubations,
performed in triplicate, were terminated by addition of
approximately 1 volume (relative to the total reaction volume) of
methanol. The samples were extracted and analyzed by LC-MS/MS, as
described above. The rates of colchicine depletion by the CYP were
compared depletion by a control for native activity (microsomes
expressing no recombinant CYP450 enzyme).
[0246] An additional control for each isoform included verification
that the isoform was active by incubating mixtures with the
universal CYP substrate, phenanthrene, and monitoring turnover of
phenanthrene fluorometrically at 254 nm (excitation) and 378 nm
(emission). The universal CYP450 positive control substrate
phenanthrene, examined with each recombinant enzyme to ensure
proper activity, confirmed that each recombinant enzyme microsomal
preparation was active (data not shown).
[0247] The results measuring colchicine disappearance at either 43
or 430 nM in incubations containing either 5 or 20 pmol of a
recombinant cytochrome P450 isozyme is summarized below in Tables
33-34.
TABLE-US-00036 TABLE 33 Metabolism of Colchicine (43 nM) by
Expressed Recombinant Human Cytochromes P450 at 5 or 20 pmol 5 pmol
cyp % Turnover Std 20 pmol cyp % Turnover 43 nM col [col], nM Mean
Std dev Mean Error [col], nM Mean Std dev Mean Std Error control
54.6 54.6 control 55.3 55.5 1.01 55.3 55.5 1.01 control 56.6 56.6
1A2 56.9 55.1 1.70 57.1 55.2 1.72 1A2 53.5 53.7 1A2 55.0 0.68 1.77
54.9 0.46 1.81 2A6 55.1 54.5 3.39 54.9 53.6 1.68 2A6 57.5 54.2 2A6
50.8 1.8 3.5 51.7 3.4 1.7 2B6 54.0 54.6 1.16 48.9 48.1 0.917 2B6
55.9 47.1 2B6 53.8 1.7 1.2 48.3 13.3 1.0 2C8 57.6 54.4 3.25 50.2
50.8 3.69 2C8 54.4 47.5 2C8 51.1 2.1 3.4 54.8 8.4 3.8 2C9 48.2 51.1
2.55 46.2 48.3 1.89 2C9 52.8 49.8 2C9 52.4 7.9 2.7 49.0 12.9 2.0
2C19 51.9 55.3 2.97 49.1 50.4 2.08 2C19 57.4 52.8 2C19 56.6 0.37
3.10 49.3 9.1 2.2 2D6 57.5 52.6 4.76 56.9 55.7 1.5o 2D6 52.3 54.0
2D6 48.0 5.2 5.0 56.1 -0.35 1.54 2E1 49.7 53.0 2.86 55.9 54.6 1.67
2E1 54.7 52.7 2E1 54.6 4.5 3.0 55.1 1.6 1.7 3A4 53.9 55.4 1.3 43.4
44.2 0.721 3A4 56.1 44.4 3A4 56.2 0.19 1.33 44.8 20.3 0.7
TABLE-US-00037 TABLE 34 Metabolism of Colchicine (430 nM) by
Expressed Recombinant Human Cytochromes P450 at 5 or 20 pmol 430 nM
5 pm cyp % Turnover Std 20 pm cyp % Turnover colchicine [col], nM
Mean Std dev Mean Error [col], nM Mean Std dev Mean Std Error
control 553 553 control 519 531 19 519 531 19 control 522 522 1A2
531 358 308 520 522 8 1A2 540 515 1A2 2.94 -0.80 0.86 530 1.9 0.8
2A6 507 500 11 500 494 13 2A6 506 479 2A6 488 5.9 1.2 502 7.1 1.4
2B6 526 528 10 510 527 15 2B6 519 536 2B6 538 0.70 1.01 534 0.91
1.56 2C8 481 489 8 485 484 7 2C8 496 490 2C8 490 7.9 0.8 477 9.0
0.7 2C9 505 505 11 525 512 14 2C9 494 497 2C9 515 5.1 1.1 515 3.6
1.5 2C19 539 520 19 505 505 7 2C19 519 498 2C19 502 2.2 2.0 512 5.0
0.8 2D6 542 535 7 500 511 12 2D6 528 523 2D6 535 -0.65 0.81 509 4.0
1.3 2E1 538 534 4 538 527 18 2E1 530 537 2E1 533 -0.41 0.45 506 1.4
1.8 3A4 538 529 12 441 438 8 3A4 515 443 3A4 533 0.52 1.29 429 17.7
0.8
[0248] Based on these experiments, several CYP450s appeared to
metabolize colchicine at 43 nM including CYP3A4, CYP2B6, CYP2C9,
and CYP2C19, which showed percent turnovers of 20.3, 13.3, 12.9,
and 9.1% at 20 pmol CYP450, and 0.19, 1.7, 7.9, and 0.37% at 5 pmol
CYP450, respectively. However, none of the enzymes showed a
statistically significant level of depletion of colchicine relative
to the control (p.ltoreq.0.05 using a t-test) in the experiments
using only 5 pmol of the CYP isozyme, although the level of
colchicine measured after exposure to CYP2C9 was almost
significantly different than the control (p=0.051). At 20 pmol CYP,
CYP2C9, CYP2C19, and CYP3A4 showed reduction in colchicine that was
significantly different from the initial colchicine present in the
control (p.ltoreq.0.05).
[0249] At 430 nM concentrations of colchicine CYP3A4, CYP2C8,
CYP2A6, CYP2C9, and CYP2C19 were most effective with percent
turnovers of 17.7, 9.0, 7.1, 3.6, and 5.0% at 20 pmol CYP450, and
0.52, 7.9, 5.9, 5.1, and 2.2% at 5 pmol CYP450, respectively.
CYP2D6 also moderately metabolized 430 nM colchicine (maximum
turnover of 3.96%). However, when the amount of colchicine detected
in the metabolism samples were compared with the amount of
colchicine detected in the control using a t-test to determine if
the two were statistically different, only CYP2C8 had a
p.ltoreq.0.05 at both 5 and 20 pmol CYP. CYP2A6 and CYP3A4 showed
statistically significant colchicine loss only at 20 pmol CYP.
[0250] Recitation of ranges of values are merely intended to serve
as a shorthand method of referring individually to each separate
value falling within the range, unless otherwise indicated herein,
and each separate value is incorporated into the specification as
if it were individually recited herein. The endpoints of all ranges
are included within the range and independently combinable.
[0251] All methods described herein can be performed in a suitable
order unless otherwise indicated herein or otherwise clearly
contradicted by context. The use of any and all examples, or
exemplary language (e.g., "such as"), is intended merely to better
illustrate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention as used
herein. Unless defined otherwise, technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs.
[0252] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *